© 1995 American Heart Association, Inc.
Articles |
Immunosuppressive Agents and Endothelial Repair
Prednisolone Delays Migration and Cytoskeletal Rearrangement in Wounded Porcine Aortic Monolayers
Presented in part at the Xth Scientific Sessions of the American College of Cardiology, Dallas, Tex, March 6-9, 1992, and published in abstract form in J Am Coll Cardiol. 1992;19:121A.
From the Vascular Research Laboratory, Department of Pathology, and The Banting and Best Diabetes Center, University of Toronto, and The Toronto Hospital, Toronto, Ontario, Canada (A.R., A.I.G.), and the UCLA Division of Cardiology, Los Angeles, Calif (A.I.F.).
Correspondence to Alistair I. Fyfe, UCLA Division of Cardiology, 47-123 CHS, 10833 Le Conte Ave, Los Angeles, CA 90024-1679. E-mail afyfe@medicine.medsch.ucla.edu.
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Abstract |
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Abstract Endothelial denudation at areas of predilection to atherosclerosis is balanced by an active repair process that may be inhibited under conditions of accelerated atherosclerosis. After cardiac transplantation, the accelerated atherosclerotic process that develops may be enhanced by immunosuppressive agents that have nonspecific effects on cell signaling, proliferation, and response to injury. To study subtle effects of cyclosporine A, azathioprine, and 6
-methylprednisolone on normal endothelial repair
processes, confluent porcine endothelial monolayers
were denuded in the presence of clinically relevant concentrations of
these agents. The rate of endothelial wound repair was
compared and the effects on cell spreading, proliferation, and the
cytoskeleton assessed. 6-Methylprednisolone at concentrations of
1.25 to 50 µmol/L was associated with a transient 30% to 60%
inhibition of endothelial wound repair. This was
associated with increased cell size at the wound edge and a delay in
centrosomal reorientation toward the wound, without any effect on cell
proliferation. Cyclosporine and azathioprine in clinically
relevant concentrations did not affect endothelial
repair. Thus, corticosteroids transiently inhibit
endothelial cytoskeletal alterations that are important
in endothelial repair after a denuding injury.
Key Words: cyclosporine A • wound repair • azathioprine • endothelium • corticosteroids
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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A central feature of atherosclerosis is the presence of a functional or structural endothelial "injury,"1 and the rate of development of atherosclerosis has been linked to the amount of endothelial injury.2 Transplant coronary disease after cardiac transplantation is the most frequent cause of death after the first year of survival.3 The diffuse nature of the disease, which is confined to the grafted aorta and coronary arteries, suggests an immunologic mechanism for endothelial injury. Prognostic factors for the development of graft atherosclerosis include repeated episodes of tissue rejection, cytomegalovirus infection,4 hypercholesterolemia,5 ischemia/reperfusion, and cardioplegia, any or all of which may contribute to endothelial injury.6 Evidence for endothelial injury in humans is derived from studies of endothelial function, which is abnormal after cardiac transplantation,7 and from observations of endothelial denudation in animal models of cardiac transplantation.8 Endothelial turnover is known to be increased in areas of predilection for atherosclerosis,9 and the loss of endothelial integrity may contribute to the migration and trapping of lipoproteins10 and monocytes11 in the subendothelial space. It follows that the ability of the endothelium to repair denuded areas may be important in the prevention of atherosclerosis in general and in the development of transplant coronary disease in particular.
Immunosuppressive agents have metabolic and cytotoxic side
effects that may contribute to atherosclerosis,
independent of their effects on the immune system. CsA binds to the
cytoplasmic protein cyclophilin and acts through a
calmodulin-dependent serine-threonine phosphatase to
prevent phosphorylation of the inhibitor of
nuclear factor–
B (I-B), a ubiquitous cytoplasmic nuclear
transcription factor.12 The immunosuppressive effect of
CsA is a result of transcriptional inhibition of interleukin-2
production.12 CsA has been shown to inhibit
EC13 and smooth muscle cell14 proliferation
in vitro but is paradoxically associated with an increase in
atherosclerosis in mice.15 Azathioprine
and its active metabolite 6-mercaptopurine are competitive
inhibitors of purine metabolism and have
preferential effects on proliferating cells. The major effect of
glucocorticoids in inflammation and immunity is to prevent the
expression of proinflammatory16 and
cytokine17 genes. Whereas glucocorticoids have
been shown to increase the size of ECs in vitro,18 recent
clinical evidence suggests that corticosteroid use is
associated with an increase in fatal cardiovascular
events.19
This study was undertaken to assess the effects of currently used
immunosuppressive drugs (CsA, azathioprine, and
6-methylprednisolone) on endothelial repair by using
a well-characterized porcine endothelium monolayer
wound model.20 The effects of these agents on cell size,
shape, proliferation, and cytoskeleton were also studied. We can show
that methylprednisolone transiently inhibits repair, actin filament
resorption, and centrosomal movement in response to
injury.
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Methods |
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Drug Preparations
The metabolically active derivative of prednisone, 6
-methylprednisolone (Sigma Chemical Co), was dissolved in 95%
(vol/vol) ethanol to 1 mg/mL. Various dilutions from 1.25 to 50
µmol/L were added directly to the culture medium before use. Control
monolayers were treated with similar concentrations of ethanol. CsA
(Sandoz Pharmaceuticals) was dissolved in absolute ethanol and Tween 80
(20 µL/mL) at 10 mg/mL. Dilutions of 100 to 3200 ng/mL were made in
standard culture medium at the time of use. Controls were treated with
ethanol and Tween 80 at similar dilutions. CsA concentrations were
measured in the culture media by high-performance liquid
chromatography to confirm the final concentrations.
Azathioprine and its active metabolite 6-mercaptopurine (Sigma) were
dissolved in 1 mol/L NH4OH (to yield a concentration of 10
mg/mL) and diluted in M199 (Whittaker) to yield a concentration of 100
µg/mL. Dilutions of 25 to 100 ng/mL were made directly into the
culture medium. Controls were treated with NH4OH at
identical concentrations.
EC Cultures
Porcine ECs were harvested as previously
described.21 In brief, porcine aortas were collected in
PBS supplemented with antibiotics, and ECs were harvested by
collagenase dispersion. Monolayers were incubated at 37°C
in a humid atmosphere containing 5% CO2. Confluent primary
cultures were passaged by trypsin-EDTA dispersion. Cells from the
fourth and eighth passages were used.
Monolayers were established by plating 1x104 cells on 22x22-mm coverslips in 35-mm tissue-culture dishes. After allowing the cells to adhere to the dishes for 24 hours, the cultures were exposed to single drugs or drug combinations with appropriate controls. Confluent monolayers were wounded by using a spatula, and three scratches, perpendicular to the wound, were made with a diamond pencil.17 The monolayers were washed twice with PBS, and medium containing the same drug combination as before wounding was added. The size of the wound was measured by phase-contrast microscopy and a x10 graduated eyepiece at 0, 24, and 48 hours at sites opposite the perpendicular scratches. Migration of the wound edge was assessed by subtracting wound size at 24 or 48 hours from baseline (0 hour). Each treatment group consisted of three sets of experiments, each with three coverslips and each with triplicate measurements (total of 27). Endothelial wound repair was expressed as mean±SD of the distance covered by the advancing wound edges in 24 hours. Comparisons among groups were made by ANOVA.
Cytoskeletal Studies
To characterize the effects of the drugs on the distribution of
actin microfilaments, microtubules, and intermediate filaments in the
cells, monolayers were washed at 24 hours in PBS, fixed with 3%
(vol/vol) paraformaldehyde,
permeabilized with 0.1% (vol/vol) Triton X-100, and
stained with fluorescence-labeled antibodies to vimentin and
tubulin as previously described.10 F-actin was stained
with rhodamine-phalloidin.10 Slides were examined by
fluorescence microscopy (Zeiss) and photographed on Kodak Tri-X
pan film. Tubulin-stained cells were examined for centrosome position
and scored as being toward the wound edge, away from the wound edge, or
in the middle, as previously described.17 19 Differences
in orientation were compared by Fisher's Exact Test.
To measure the size of single cells at the wound edge, photomicrographs
of hematoxylin and eosin–stained monolayers were analyzed
quantitatively using Apple computer software and a graphics tablet. The
cell size was calculated for 20 cells from control cultures and 20
cells from 6-methylprednisolone (20 µmol/L)–treated cultures.
Differences in cell size were compared by Student's t
test.
Cell Proliferation Studies
The effect of 6-methylprednisolone (2.5 and 20 µmol/L) on
EC proliferation was assessed by BrdU incorporation into cellular DNA
24 hours after wounding. Cultures were incubated for 1.5 hours with a
BrdU labeling reagent (Amersham) at 37°C. After a brief wash in PBS
the cultures were treated with acetone at 4°C for 10 minutes and then
fixed with ethanol/acetone (95/5, vol/vol) for 20 minutes. The cultures
were washed in PBS and incubated with BrdU antibody for 1 hour at room
temperature, followed by an incubation with rabbit anti-tubulin
antibody (ICN) for 30 minutes. After being washed in PBS, the cultures
were incubated with donkey anti-rabbit IgG–Texas red (Sigma) to
localize tubulin and to define the number of cells present. Cell
fluorescence was observed with a confocal laser microscope
(Bio-Rad MRC-600) set for detecting both FITC and Texas red. Cells in
the first eight rows from the wound edge were counted for BrdU
incorporation. Results were expressed as a proportion of the total cell
population, and comparisons were made by Student's t
test.
The effect of 6-methylprednisolone on cell proliferation was also
studied in low-density culture. ECs were grown in 60-mm dishes in M199
(Whittaker) and divided into three groups (controls without drug or
ethanol, 6-methylprednisolone at 2.5 or 20 µmol/L, and controls
with ethanol). The drug or ethanol was added 24 hours after plating. At
confluence, cultures were trypsinized and 2x104 cells were
replated in 35-mm culture dishes in M199 (Whittaker) to assess cell
growth in the presence of 6-methylprednisolone or ethanol. Cell
counts were performed using a Coulter counter on trypsinized plates 18
and 42 hours after plating. Results were expressed as the mean of three
dishes for each time point, and comparisons were made by Student's
t test.
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Results |
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At the highest concentration of CsA (1600 ng/mL), confluence of the endothelial monolayer was delayed by 4 to 6 days and thus, wounding could not be performed. There was no significant inhibition of endothelial repair in the first 24 hours at CsA concentrations of 50 to 800 ng/mL (Table 1
).
Repair of endothelial wounds averaged 61±7 µm during
the first 24 hours after wounding and 84±10 µm during the second
24-hour period in the control cultures containing ethanol and Tween 80.
CsA concentrations of 200 and 400 ng/mL were associated with wound edge
migration of 66±10 and 67±10 µm, respectively in the first 24 hours
(P=NS). At CsA concentrations of 400 and 800 ng/mL, there
was significant inhibition of healing in the second 24-hour period
(Table 1).
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Endothelial wound repair in monolayers exposed to
6-mercaptopurine was comparable to that of control cultures: migration
during the first 24-hour period was 59±9 µm for controls, 63±11
µm for a 6-mercaptopurine concentration of 25 ng/mL, and 63±10 µm
for a 6-mercaptopurine concentration of 50 ng/mL. There were no
differences in the 24-hour wound healing rates in
endothelial cultures exposed to 25 to 50 ng/mL
azathioprine compared with those of 6-mercaptopurine–treated cells
(data not shown). There was an incremental dose-dependent inhibition of
endothelial wound repair during the first 24 hours in
response to 6-methylprednisolone (Table 1
and Fig 1).
Even at the lowest dose tested (1.25 µmol/L, which corresponds to a
dose of 0.5 mg/kg body weight), there was a significant reduction in
wound repair at 24 hours (58±10 versus 77±2 µm,
P<.005). However, this effect was lost in the second
24-hour period, suggesting that either the 6-methylprednisolone had
been metabolized or the ECs had overcome blockade. Readdition of
6-methylprednisolone at 24 hours significantly inhibited wound
repair at 24 to 48 hours, from 73±14 to 37±8 µm
(P<.005). The addition of 6-methylprednisolone to 200
ng/mL of CsA and to 50 ng/mL 6-mercaptopurine demonstrated no synergism
between the drugs when compared with 6-methylprednisolone alone (Fig 2).
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The delay in endothelial wound repair could be a result
of a decrease in cell spreading, a delay in migration, or inhibition of
endothelial proliferation, as the wound size was
sufficient to require all three processes for closure. The effect of
6-methylprednisolone on cell spreading was assessed by measuring EC
size at the wound edge. 6-Methylprednisolone–treated cells were, on
average, 22% larger than control cells (4.6±0.8 versus 3.7±0.8
10-3 mm2, P<.005).
Immunofluorescence staining was used to assess drug
effects on vimentin, tubulin, and actin cytoskeletal responses to
injury in the endothelial monolayers. There were no
changes in actin, vimentin, or tubulin after exposure to CsA,
azathioprine, or 6-mercaptopurine. In monolayers treated with
6-methylprednisolone, the dense, peripheral band of
actin, which normally dissolves before the initiation of cellular
migration, persisted 24 hours after wounding when compared with
untreated monolayers. This was particularly obvious in rows 6 through
10 from the wound edge (Fig 3).
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Immunofluorescence staining for -tubulin
was used to assess microtubule organization and centrosome location.
The orientation of the centrosome from its resting orientation, ie,
random with respect to the wound edge, is a critical event in directing
the migration of the monolayer in response to injury.17 At
24 hours the proportion of centrosomes that had reoriented toward the
wound edge was significantly reduced in
6-methylprednisolone–treated monolayers (Table 2).
There were no differences in centrosome orientation after exposure to
cyclosporine, 6-mercaptopurine, or azathioprine (data not
shown).
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Finally, the effect of 6-methylprednisolone on EC proliferation was
assessed. Treatment of ECs with 2.5 or 20 µmol/L 6-methylprednisolone
enhanced the incorporation of BrdU into the DNA of cells in the first 8
rows from the wound edge. In monolayers exposed to 2.5 µmol/L
6-methylprednisolone, the proportion of proliferating cells was
58.0±4.3% compared with 47.9±1.7% (P<.02) for the
controls. At 20 µmol/L 6-methylprednisolone, 62.8±1.6% of the
treated cells were proliferating compared with only 46.8±0.7%
(P<.01) of the control cells. In contrast, treatment of
low-density ECs with 6-methylprednisolone did not have any significant
effect when cell proliferation was measured 42 hours after plating
(data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The repair of endothelial wounds in vitro requires cell spreading if the wound is small22 and spreading, migration, and proliferation20 23 if the wound is large. In this series of experiments, we sought to test the hypothesis that immunosuppressive agents alone or in combination might inhibit the repair of wounded endothelial monolayers. A lack of healing of recurrent endothelial injury, which may be immunologic, could be a potential mechanism for the accelerated atherosclerosis that is commonly seen after transplantation.6 This would be consistent with data that have shown increased endothelial turnover in areas of predilection for spontaneous atherosclerosis.9 Previous studies have shown a specific sequence of cytoskeletal events that occur during the process of endothelial wound repair in vitro20 22 23 and in vivo.24 Within seconds of creating a wound in an endothelial monolayer, an influx of calcium is noted in adjacent cells.25 Centrosomes have been shown to translocate toward the wound edge as early as 10 seconds after wounding26 and certainly within 1 to 2 hours.22 23 24 The cells spread toward the wound by extension of their lamellipodia but without translocation, and the dense, peripheral band of actin microfilaments remains intact.23 If the wound remains open for longer than 1 hour, the cell undergoes a further series of changes.20 The dense, peripheral actin band is then disassembled, and the cell elongates and migrates toward the wound edge.
In these experiments 6-methylprednisolone produced a
significant, transient, dose-dependent inhibition of repair in this
injured monolayer model, whereas CsA and 6-mercaptopurine had no
effect. 6-Methylprednisolone was used in this system because it is
the metabolically active derivative of prednisone, the most
commonly prescribed corticosteroid. Orally administered
doses of 1 mg/kg body weight would correspond to serum prednisolone
concentrations of 2.6 µmol/L if one assumes 100% conversion by the
liver. The first response to endothelial injury is cell
spreading,22 and a significant increase in EC size was
noted in 6-methylprednisolone–treated cultures. A similar increase
in EC size was not seen when the metabolically inactive
parent compound prednisone was tested.18 An increase in
cell size should favor wound closure, and the marked reduction in
endothelial wound repair in
6-methylprednisolone-treated monolayers suggested that the effect
must be on migration or proliferation. Studies using BrdU incorporation
in the monolayer or measurement of proliferation in low-density
cultures showed either no effect or a significant increase in the
proliferation of 6-methylprednisolone–treated ECs, suggesting that
the major effect of corticosteroids on
endothelial healing is on cell migration.
In endothelial monolayers, the presence of a wound edge, even when migration is inhibited, is enough to induce centrosome relocation.20 Centrosome redistribution precedes other cytoskeletal events in wound repair20 22 and requires intact microtubules24 and microtubule-microfilament interactions.27 Inhibition of transcription in wounded endothelial monolayers prevents both centrosome formation and wound repair.28 Cell migration requires not only active actin disassembly, which is triggered by calcium entry into the cell, but also transcription of specific genes.29 Cytoskeletal studies showed that 6-methylprednisolone transiently inhibited the formation and relocation of centrosomes toward the wound edge and delayed resorption of the dense, peripheral band of actin. It is interesting to speculate that glucocorticoids prevent the expression of specific genes that are involved in cytoskeletal rearrangement. Glucocorticoids block expression of other early-response and cytokine genes, either by inhibiting transcription or destabilizing mRNA.16 17 Migration, proliferation, and differentiation of ECs are important steps in angiogenesis, which is also inhibited by glucocorticoids.30 Glucocorticoids also inhibit the migration of retinal pigment ECs in vivo.31
Clinical and experimental evidence has suggested a correlation between corticosteroid use and the development of accelerated atherosclerosis. Exposure to corticosteroids after cardiac transplantation is associated with increased intimal thickness as measured by intravascular ultrasound,32 and the combination of increased cellular rejection and corticosteroids (ie, more injury and inhibition of repair) is associated with fulminant transplant coronary disease.33 There is further evidence to suggest that steroid-free immunosuppressive regimens after cardiac transplantation are associated with a lower incidence of transplant atherosclerosis.34 The use of corticosteroids in the treatment of rheumatic arthritis was recently found to be an independent risk factor for fatal cardiovascular events.19 In the setting of coronary artery angioplasty, restenosis was not prevented when methylprednisolone was used either as a single dose or on alternate days,35 suggesting, as noted in our in vitro culture, that the short half-life of this drug may be important in determining its ability to prevent cell migration. In vivo, a secondary effect of corticosteroid therapy, that of hyperglycemia, may also be active in preventing endothelial healing.36
Significant differences in endothelial responses to cyclosporine appear to depend on their vascular origin. Human umbilical vein EC proliferation is inhibited at cyclosporine concentrations greater than 625 ng/mL12 and rat microvascular EC growth is inhibited at 250 ng/mL,13 but rabbit aortic ECs are not inhibited at 5000 ng/mL.37 The inhibition of EC growth at 1600 ng/mL noted in our experiments is consistent with these studies. Inhibition of endothelial repair in the first 24 hours was not seen in our studies at CsA concentrations up to 800 ng/mL, as measured by high-performance liquid chromatography of the culture media. The small decreases in wound closure in the second 24-hour period at higher doses of CsA may reflect inhibition of proliferation, as this is the predominant response at this time point after endothelial wounding.28 There were no measurable effects on endothelial wound repair from azathioprine or 6-mercaptopurine at clinically relevant concentrations.38
In summary, in this porcine arterial
endothelial monolayer model, 6-methylprednisolone
transiently inhibited endothelial wound repair by
delaying migration and cytoskeletal rearrangement. CsA showed
significantly less inhibition of wound closure only 24 to 48 hours
after wounding. The known predilection of
atherosclerosis for sites of
endothelial denudation and turnover would imply that
any delay in the formation of a confluent endothelial
monolayer may be pathophysiologically related
to the development of atherosclerosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by Medical Research Council of Canada grant MI-6485 (to Dr Gotlieb) and Heart and Stroke Foundation of Ontario grant 1964 (to Dr Gotlieb). We thank S. Sarju for secretarial support.
Received February 12, 1993; accepted May 17, 1995.
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