Circulation Research. 2000;86:334-340
(Circulation Research. 2000;86:334.)
© 2000 American Heart Association, Inc.
Photodynamic Therapy Generates a Matrix Barrier to Invasive Vascular Cell Migration
Marcus Overhaus,
Joerg Heckenkamp,
Sylvie Kossodo,
Dariusz Leszczynski,
Glenn M. LaMuraglia
From the Division of Vascular Surgery (M.O., J.H., G.M.L.) and the
Wellman Laboratories of Photomedicine (M.O., J.H., S.K., D.L., G.M.L.),
Massachusetts General Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Glenn M. LaMuraglia, Massachusetts General Hospital, Department of Surgery, Fruit Street, Boston, MA 02114.
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Abstract
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AbstractPhotodynamic therapy
(PDT) inhibits experimental
intimal hyperplasia. PDT results in
complete vascular wall cell
eradication with subsequent adventitia but
minimal media repopulation.
This study was designed to test the
hypothesis that PDT alters
the vascular wall matrix thereby inhibiting
invasive cell migration,
and as such, provides an important barrier
mechanism to favorably
alter the vascular injury response. Untreated
smooth muscle
cells (SMCs) and fibroblasts were seeded on control and
PDT-treated
(100 J/cm
2; photosensitizer was
chloroaluminum-sulfonated phthalocyanine,
5 µg/mL) 3-dimensional
collagen matrix gels. Invasive
cell migration was temporally quantified
by calibrated microscopy.
Zymography and ELISA assessed SMC matrix
metalloproteinase levels.
Molecular changes of gel proteins and their
susceptibility to
collagenase were analyzed by
SDS-PAGE and Western blot. Limited
pepsin digestion and histology were
used to assess the in vivo
relevance of the model, using an established
rat carotid artery
model at 1 and 4 weeks after balloon injury and PDT.
PDT of
3-dimensional matrix of gels led to a 52% reduction of invasive
SMCs
and to a 59% reduction of fibroblast migration
(
P<0.001)
but did not significantly affect secretion of
matrix metalloproteinases.
PDT induced collagen matrix changes,
including cross-linking,
which resulted in resistance to protease
digestion. PDT led
to a durable 45% reduction in pepsin digestion
susceptibility
of treated arteries (
P<0.001) and
inhibition of periadventitial
cell migration into the media. These data
suggest that PDT of
matrix gels generates a barrier to invasive
cellular migration.
This newly identified effect on matrix proteins
underscores
its pleiotropic actions on the vessel wall, and as such,
PDT
may be of considerable potential therapeutic value to inhibit
restenosis.
Key Words: photodynamic therapy restenosis cell migration collagen metalloproteinases
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Introduction
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Restenosis remains the major obstacle to
long-term success after
invasive vascular procedures.
1 As
part of the injury to the
vessel wall, vascular smooth muscle cells
(SMCs), adventitial
myofibroblasts, and fibroblasts proliferate and
migrate into
the subintimal space, where they deposit extracellular
matrix
(ECM), thereby resulting in lumen loss.
2 3 The role
of cellular
migration from the media and the adventitia has remained a
focal
point in the effort to identify an approach to inhibit vascular
restenosis.
Using this strategy, inhibitors of
matrix metalloproteinases
(MMPs), enzymes crucial for invasive SMC
migration,
4 ECM repair,
and remodeling,
5
prevented SMC migration, thus resulting in
a temporary repression of
experimental intimal hyperplasia (IH).
6 This transient
success reflects the complexity of vascular
injury and subsequent
healing and emphasizes why restenosis
is a difficult problem to
solve. Of the many approaches tested,
only stents
7 and
ionizing irradiation
8 have been clinically
demonstrated to
reduce this process.
Photodynamic therapy (PDT) is another promising approach undergoing an
early clinical trial.9 PDT uses light to activate
otherwise inert photosensitizer dyes to produce photochemical reactions
through the production of free radical moieties without the
generation of heat.10 These free radicals eradicate the
entire cell population of the artery wall without inducing inflammation
or structural deterioration and thus result in long-term inhibition of
experimental IH.11 12 Vascular PDT has other effects,
including inactivation of matrix-associated cytokines and
growth factors, which result in alteration of vascular cell
function.13 These matrix effects may influence the
observed cellular repopulation of PDT-treated arteries, including
reendothelialization and repopulation of the
adventitia, but delayed and only sparse repopulation of the
media.12
This study tests the hypothesis that PDT generates a matrix barrier to
cell migration through the vessel wall. This barrier would inhibit
cells from the adventitia from migrating into the intima, therefore
explaining, in part, the favorable effects of PDT in vivo. To this end,
SMC and fibroblast-invasive migration through control and PDT-treated
3-dimensional (3D) collagen matrix gels were studied. In addition, the
mechanisms by which PDT-altered matrix inhibited SMC migration were
investigated. To accomplish this, MMP levels were assessed in cultures
of SMCs on PDT-treated matrix gels, and changes of the molecular
structure of the matrix after PDT were determined. To demonstrate the
in vivo relevance of these data, we investigated the levels and
stability of PDT-induced cross-links in the rat carotid artery and its
effect on cellular migration through the artery after balloon injury
and PDT.
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Materials and Methods
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In Vitro PDT Effects on Collagen Type I Cross-Linking and
Inhibition of Cellular Migration
Cell Culture
Bovine aortic SMCs and adventitial fibroblasts were obtained
and
cultured as described before.
14 15
3D Collagen Matrix Gel
3D collagen type I matrix gels
(Vitrogen100 Collagen Biomaterials) were prepared
as described,16 and for PDT or photosensitizer-only
control, chloroaluminum-sulfonated phthalocyanine (5 µg/mL, Novartis)
was added. Control groups included albumin instead of calf
serum (CS), to control for growth factor-independent migration, and
D-ribose (4 mg/mL, 4 days), known to induce
glycation-dependent cross-links.17
PDT Treatment
After gelation, the matrix gels were irradiated with in vivo
effective laser light dosimetry (100 J/cm2; 100
mW/cm2;
=660 nm).
Cell Migration Assay
Untreated SMCs and fibroblasts were seeded on the matrix gel
surface at 8x104 cells/well. Migration was
assessed at 4, 8, and 11 days18 by calibrated
phase-contrast microscopy (Zeiss IM35). Cells were counted at 0.8-mm
depth and in a cylindrical field through the entire depth of the gels.
SMC morphology was assessed at x400 magnification.
Zymography
Gelatin zymography assessed MMP-2 and MMP-9 secreted by SMCs at
day 8. SDSpolyacrylamide gels with copolymerized 0.2%
gelatin (Sigma) were used.19
ELISA
No specific test for bovine MMP-1 is available. MMP-1 levels
were determined using a human MMP-1 ELISA (Calbiochem), given that
human and bovine MMP-1 are 87% homologous.20 This assay
does not cross-react with MMP-2, MMP-3, or MMP-9.
Gel Electrophoresis
The resistance of collagen type I matrix gel solution (1.5
mg/mL) to collagenase was assessed by digesting matrix gel
solutions with clostridial collagenase (600 to 1200
µg/mL; Gibco) followed by SDS-PAGE (5%).21
Western Blot
Collagen matrix changes were revealed with rabbit anti-bovine
collagen type I antibody (diluted 1:160, Biodesign), followed by an
anti-rabbit IgG (diluted 1:1000).
In Vivo PDT Effects on Matrix Cross-Linking and Cellular
Migration
Animal Model
Animal care was in compliance with Principles of
Laboratory Animal Care and the Guide for the Care and Use of
Laboratory Animals (NIH publication No. 80-23, revised 1985) and
approved by the institutional animal care committee.
Rats (Charles River) were anesthetized with ketamine
(35 mg/kg), atropine (40 µg/kg), and xylazine (5 mg/kg).
Photosensitizer application, balloon injury of the carotid artery, and
laser irradiation were performed as described.12 Animals
were euthanized at 1 hour (n=3) and at 1 (n=3) and 4 (n=3) weeks after
PDT (n=3).
Histology
To assess cell migration after 1 and 4 weeks, the PDT-treated
artery was fixed with formalin, and cross-sections were stained with
hematoxylin and eosin for light microscopy.
Limited Pepsin Digestion
To determine matrix cross-linking, the unfixed artery and the
untreated contralateral artery were harvested for pepsin
digestion.22
Statistical Analysis
Results were expressed as mean±SD. A 1-way ANOVA and Tukey post
hoc test were applied. Differences between controls and PDT in
zymography, ELISA, and pepsin digestion experiments were
analyzed with the t test for independent
variables by means of the Statistica software (Statsoft). A
P-value <0.05 was considered significant.
An expanded Materials and Methods section is available online at
http://www.circresaha.org.
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Results
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In Vitro PDT Effects
Cell Migration Assay
Migration of untreated SMCs and fibroblasts was assayed on control
and
PDT-treated matrix gels. In all groups, cells formed a homogenous
monolayer
on the 3D gel surface within 48 hours. PDT of the matrix gel
decreased
the total number of SMCs that migrated into the 3D gel by
52%
at 11 days (
P<0.001, Figure 1A

). In addition, the depth of
migration
was significantly reduced in PDT-treated gels (61%
reduction,
P<0.01, Figure 1B

). PDT also decreased the total
number
of fibroblasts that migrated into the 3D gel by 59% at 11 days
(
P<0.001,
Figure 1C

), as well as the depth of
migration (77% reduction,
n=15,
P<0.001, Figure 1D

).

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Figure 1. SMC and fibroblast migration into PDT-treated,
control, and D-ribosetreated matrix gels. Data are
mean±SD. A, Total SMC number migrating into matrix gels per
microscopic field. Total SMC migration was measured after 4, 8, and 11
days. *P<0.001 at all time points in PDT vs controls vs
D-ribose.
n=15 for all groups. B, SMC number migrating into the
matrix gels at a specific depth (0.8 mm) per microscopic field.
Total SMC number at 0.8-mm depth was measured after 4, 8, and 11 days.
*P<0.01 at all time points in PDT vs controls vs
D-ribose. n=15 for all groups. C, Fibroblast migration
(total number). Total fibroblast number migrating into matrix gels per
microscopic field. *P<0.001 at all time points in PDT
vs controls vs D-ribose. n=15 for all groups. D, Fibroblast
number migrating into the matrix gels at a specific depth (0.8 mm)
per microscopic field. *P<0.001 in PDT vs controls vs
D-ribose; +P<0.01, D-ribose vs
controls at 4 days. n=15 for all groups. E, Dose-dependent SMC
migration (total number). Total SMC number migrating into matrix gels
per microscopic field. *P<0.001 at 11 days, 50
J/cm2 vs 100 J/cm2; +P<0.05 at 11
days, 100 J/cm2 vs 200 J/cm2. n=15 for
all groups.
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Adding D-ribose, known to induce glycation-dependent
protein cross-links to the 3D gel, decreased cell migration of both
cell types similarly to the decrease observed in PDT-treated
gels. Cells on control gels (laser-only, photosensitizer-only, and
albumin instead of CS matrix gel) had migration patterns
similar to those on untreated gels (data not shown).
Invasive SMC migration was PDT dose-dependent. At 50
J/cm2, the total number of cells migrating into
the gel at 11 days was 33% higher compared with 100
J/cm2 (P<0.001) and at 100
J/cm2, 20% higher compared with 200
J/cm2 (P<0.05, Figure 1E
).
Vascular Cell Morphology
SMC morphology at the surface of control and PDT-treated matrix
gels did not differ. Cells were flat with thin, well-spread filopodia
following the main cell axis, adopting a stellate shape. Cells
migrating into control gels were similar, whereas cells migrating into
PDT-treated gels appeared cylindrical with an apparently reduced
cytoplasm, loss of the stellate shape, and decreased spreading
filopodia (Figure 2
). Fibroblasts
migrating into PDT-treated gels showed a similar change in morphology
(data not shown).

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Figure 2. Photomicrograph of SMCs in matrix gels
(phase-contrast microscopy). Upper panel represents SMCs in
untreated gels; lower panel, SMCs in PDT-treated gels. Original
magnification, x400. Bar=5 µm.
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Zymography
Zymography determined the levels of MMP-2 and MMP-9 secreted by
SMCs cultured in the differently treated matrix gels (Figure 3
). Densitometry analysis
revealed no significant differences (P=0.7) in MMP levels
between SMCs cultured on untreated or PDT-treated matrix gel.

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Figure 3. Detection of MMP-2 and MMP-9 by gelatin
zymography. Conditioned media from cultured SMCs 8 days after seeding
on untreated (lane A) and PDT-treated (lane B) matrix gels were run on
a gelatin zymography. CS only served as a positive control (lane C).
Area of white bands indicates metalloproteinase digestion. MMP-9 and
MMP-2 are shown as latent (Pro-MMPs) and activated (MMPs)
forms.
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ELISA
An ELISA was performed to assess differences in the levels of
MMP-1, which specifically degrades collagen type I. SMCs plated on
PDT-treated gels had higher MMP-1 levels compared with SMCs plated on
control matrix gels (106.16±4.9 versus 91.04±2.6 ng/mL, n=6,
P=0.06).
Gel Electrophoresis
Control and PDT-treated matrix gel solutions (collagen type I,
10% CS, and DMEM) were analyzed by SDS-PAGE to detect
molecular weight differences (Figure 4
).
Analysis revealed new high molecular weight protein oligomers
in the stacking gel. In addition, new bands in the
-chain range with
molecular mass from 170 to 250 kDa were detected after
PDT. Furthermore, 2 new protein bands migrated below the
ß1,2 and ß1,1 band, and
the original ß1,2 band disappeared. An
additional protein band was found in the PDT-treated samples above the
1 chain, and bands below the
chains fused
into a single band (Figure 4A
). Densitometry analysis
confirmed the generation of proteins with different molecular weights
in PDT-treated 3D gels (Figure 4B
). In addition, SDS-PAGE was
performed to detect specific cross-links in PDT-treated matrix
solutions containing collagen alone, albumin alone, or both
collagen and albumin. SDS-PAGE of PDT-treated collagen solution
alone showed a distinct band of high molecular weight in the stacking
gel and a faded band above the
chain, with a loss of all lower
molecular weight bands seen in controls (Figure 4C
, lanes A and
B). Albumin alone presented the typical albumin
band without changes after PDT (Figure 4C
, lanes E and F).
Solutions containing both collagen and albumin showed a shift
from lower to higher molecular weight after PDT with loss of the
original bands and a new distinct band in the stacking gel (Figure 4C
, lanes C and D). Resistance of control and PDT-treated
collagen matrix solutions to digestion were investigated after
incubation with collagenase. In the control groups, all
higher molecular weight bands and part of the lower molecular weight
bands were digested in a dose-dependent fashion, whereas PDT treatment
resulted in resistance to digestion at all doses of
collagenase used (Figure 4D
).

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Figure 4. Gel electrophoresis of PDT-treated and untreated
matrix gel solutions (10% CS and media). A, SDS-PAGE. A lanes,
Untreated matrix gel solutions. B lanes, PDT-treated matrix gel
solutions. B, Densitometry analysis of PDT-treated and control
collagen type I solution (10% CS and media). Molecular weight on the
x axis is plotted against density on the y axis. Positions of different chains of
PDT-treated compared with untreated collagen type I solution are
indicated as , ß, and chains. C, SDS-PAGE. A lane, Collagen
type I untreated. B lane, Collagen type I PDT treated. C lane, Collagen
type I and albumin untreated. D lane, Collagen type I and
albumin PDT treated. E lane, Albumin untreated. F lane,
Albumin PDT treated. D, Collagenase digestion of
PDT-treated and untreated collagen solution. A lane, Untreated collagen
solution. A1 lanes, Untreated collagen solution with
different collagenase concentrations (600 to 1200 µg/mL).
B lane, PDT-treated collagen solution. B1 lanes,
PDT-treated collagen solution with different collagenase
concentrations. Collagenase samples were incubated for 2
hours in 37°C.
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Western Blot Analysis
Western blot using an anti-collagen type I antibody to
specifically identify new bands in matrix gel solutions after PDT
confirmed that the newly generated cross-links contained collagen
(Figure 5
). Western blot analysis
with the SDS-PAGE results (Figure 4
, lanes C and D) revealed the
PDT-induced interactions between collagen and CS. This resulted in a
shift of lower (60 to 100 kDa) to higher (100 to 350 kDa) molecular
weight bands with a noticeable change to
, ß, and
chains in
controls. Nonspecific antibody binding in the low molecular weight
range appeared only in pure untreated CS, whereas after PDT, diffuse
nonspecific binding of the whole lane was observed (Figure 5
, lanes C1 and C2).

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Figure 5. Western blot for collagen type I of PDT-treated
and untreated control matrix gel solutions. A lanes, Untreated
solutions. B lanes, PDT-treated solutions. C lanes, CS (C1, untreated;
C2, PDT-treated); origin of protein bands in the SDS-PAGE. Collagen
type I was detected in all molecular weight ranges after transfer.
Newly identified complex formation after PDT treatment contained
collagen type I as 1 component. The same amount of collagen type I
(0.75 mg/mL) was loaded onto each lane.
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In Vivo PDT Effects
Limited Pepsin Digestion
PDT-treated arteries showed a significantly lower susceptibility
to pepsin digestion at all time points measured (1 hour, 44%; 1 week,
36%; 4 weeks, 44%; P<0.0001, Figure 6
).

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Figure 6. Susceptibility of PDT-treated and untreated rat
carotid arteries to pepsin digestion. Percentage of digestion; data are
expressed as mean±SD. Pepsin digestion was measured after 1 hour, 1
week, and 4 weeks. *P<0.001 at all time points in PDT
vs untreated controls. n=3 in both groups.
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Histology
No IH was found at 1 and 4 weeks after balloon injury and PDT. At
1 week, no endothelium but occasional platelets
adhering to the internal elastic lamina were present. In contrast,
at 4 weeks, the artery was reendothelialized. At both
time points, the adventitia was repopulated with cells; however, cells
were unable to migrate into the media (Figure 7
).

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Figure 7. Composite of light micrographs from
balloon-injured rat carotid arteries treated with PDT. Internal elastic
lamina (arrow) is noted. A, One week after PDT. B, Four weeks after
PDT. No IH was noted at both time points. Note the
reendothelialization after 4 weeks. The adventitia was
repopulated, but no cells were able to migrate into the media at both
time points. Bar=50 µm.
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Discussion
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In this study, PDT modified proteins in a collagen type I matrix
gel
and in an artery to create a barrier to invasive vascular cell
migration.
Evidence supports that cellular migration through the
vascular
wall into the subintimal space is a consequence of vessel
injury
and plays a critical role in the development and progression
of
IH.
23 PDT inhibits IH by acute cell eradication, which is
followed
by cell repopulation of the adventitia but not the
media.
12 Because it is hypothesized that inhibition of
cellular migration
through the vessel wall may be an important factor
in altering
the postinterventional wound-healing response, the effect
of
PDT on invasive cell migration was investigated. A 3D matrix
gel
model was established, because the interactions between
migratory cells
and the surrounding matrix are of paramount
significance in vascular
biology.
24 A matrix gel of collagen
type I was selected
because it is one of the major connective
tissue components of the
arterial wall and surrounds SMCs forming
a lattice network
within the media.
25 In addition to soluble
collagen type
I, this matrix also contains collagen type III
and partially denatured
collagen, as well as proteins deposited
by the SMCs after plating.
Because this model does not fully
reflect the composition of vascular
ECM in vivo, the relevance
of the method was compared with PDT-induced
matrix effects on
in vivo migration using a balloon-injured artery
model.
Next to SMCs, myofibroblast and fibroblasts are known to play a role in
the vascular wound-healing response.2 Because
myofibroblasts are not morphologically and functionally well
defined,26 SMCs, which take on a secretory
phenotype in vitro similar to injured SMCs in
vivo,27 and adventitial fibroblasts, were used for the in
vitro experiments. SMCs and fibroblasts themselves were not PDT
treated, so they could simulate the nontreated, adjacent cell
populations that repopulate the vessel wall after the complete cell
eradication by PDT in vivo.
Under physiological conditions, SMCs are quiescent
and embedded in ECM.28 MMPs, produced by vascular SMCs,
are upregulated after arterial wall injury and are
necessary for cell migration during the development of
IH.18 29 30 In this study, collagenase was
unable to digest the PDT-treated matrix to permit adequate migration.
However, this is not the only MMP involved in collagen type I
degradation. Other proteinases, such as MMP-2, MMP-13, and membrane
type 1 MMP have also been shown to degrade collagen type
I31 32 33 and play important roles in the migration of
vascular cells.4 In this study, activated MMP-2
was indeed present in the conditioned media, which may explain in
part why migration was significantly reduced but not abolished.
After PDT, resistance of the matrix to collagenase might
not only be important in inhibiting cellular migration from the
adventitia but also be crucial in maintaining the mechanical integrity
of the vessel wall.12 This concurs with previous findings
of protein cross-link resistance to enzymatic
digestion.34
Inhibitors of MMPs, in particular tissue
inhibitor of metalloproteinase (TIMP)-3, which is known to
be deposited into the ECM, could play an important role in modulating
the cellular repopulation of the media after PDT. However, PDT has been
demonstrated to inactivate biologically active
matrix-associated proteins.35 Therefore, it seems unlikely
that PDT could augment the biological effect of a matrix-associated
protein such as TIMP-3.
Differences in depth and number of cells migrating into PDT-treated
matrix gels were dose-dependant, emphasizing the importance of PDT
dosimetry to inhibit IH in vivo.12 Cells exhibited a
different morphology in matrix gels as compared with controls. This
change in cell morphology, in which the cells appeared to be attempting
to insinuate themselves through small spaces in the matrix gels,
supported the hypothesis that the cross-linked and otherwise altered
proteins did not permit the cells to digest the matrix with MMPs and
expand to their regular configuration. Cell shape and migration are
interconnected through the interactions between integrins and the ECM.
It is conceivable that PDT, by modifying integrin binding sites on the
matrix, not only affects the ability of the cell to migrate but also
the cytoskeletal organization of the cell.27
To further elucidate the mechanism by which PDT inhibits vascular cell
migration, changes in the structure of collagen were investigated. PDT
has been shown to alter matrix-associated proteins.35 This
effect appears to be principally mediated by free radical interactions
with amino acids, which lead to conformational and other chemical
changes that modify biologically active or specific binding sites of
these proteins. This study identified that PDT of collagen type I
generated high molecular weight complexes, suggesting cross-linking
with increased thermal and mechanical stability.36 PDT
treatment of control matrix gel solution containing collagen alone
induced distinct collagen-to-collagen cross-links. However, the protein
cross-links in this specific 3D model, which contained collagen type I
and serum, did not only involve collagen-to-collagen interactions.
Albumin, the major protein component of serum, by itself did
not form cross-links, but in the presence of collagen, it formed
heterotypic cross-links different from those noted in the pure collagen
solution. These interactions between different molecules suggest that
PDT of the vessel wall, which is composed of various proteins,
including elastin and fibronectin, can also generate homotypic and
heterotypic cross-links. The importance of these observations is
underscored by the findings that PDT of an artery resulted in a reduced
susceptibility to pepsin digestion. This suggested the in vivo
formation of cross-links similar to the in vitro matrix gel data
presented and resulting in a barrier for vascular cells to
migrate into the media. These data thus provide a novel link between
the induction of protein cross-links and the inhibition of cellular
migration in the vessel wall, thereby explaining the PDT-induced
inhibition of IH.
Invasive cellular migration, which is one of the key factors in the
vascular wound-healing response, is modulated by a variety of
mechanisms. Using this knowledge, experimental approaches to inhibit IH
by using MMP inhibitors6 or integrin binding
inhibitors37 only resulted in short-term
inhibition. These data underline the complexity of events leading to
restenosis and suggest that inhibition of cellular migration by
disrupting a single pathway may not be sufficient in ultimately
preventing restenosis. This study identified yet another PDT
effect on the vessel wall: the inhibition of vascular cell migration by
stabilization of the matrix, rendering it resistant to
collagenase degradation, and the possible alteration of
integrin binding sites.10 These newly identified PDT
matrix effects on invasive vascular cell migration, in conjunction with
other known effects on the vascular wall, such as complete inactivation
of cell- and matrix-associated cytokines and growth factors,
could all be major and necessary targets for the observed long-term
PDT-mediated inhibition of IH. Thus, because of its multiple effects,
PDT is a unique therapeutic approach for inhibiting vascular
restenosis and provides a strong theoretical basis for its
successful clinical application.
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Acknowledgments
|
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This study was supported in part by the Office of Naval Research
Contract
N00014-94-I-0927 and the Albert DiGregorio Foundation. J.H.
is
a recipient of a Deutsche Forschungsgemeinschaft fellowship
grant (He
2926/1-1). We thank Drs J. Parrish and R. Anderson
for support and Dr
Jialin Bao for his excellent technical assistance.
We acknowledge
Novartis for providing chloroaluminum-sulfonated
phthalocyanine and
Polaroid for the diode laser.
Received June 29, 1999;
accepted November 22, 1999.
 |
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