© 1997 American Heart Association, Inc.
Articles |
Inhibitory Effects of Hypercholesterolemia and Ox-LDL on Angiogenesis-like Endothelial Growth in Rabbit Aortic Explants
Essential Role of Basic Fibroblast Growth Factor
From the Departments of Medicine (C.-H.C., Z.L., S.L., H.H.N., A.M.G. Jr, P.D.H.) and Pathology (J.C. Jr), Baylor College of Medicine, Houston, Tex.
Correspondence to Chu-Huang Chen, MD, PhD, Department of Medicine, Mail Station A601, Baylor College of Medicine, 6565 Fannin, Houston, TX 77030. E-mail cchen{at}bcm.tmc.edu
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Abstract |
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Abstract Hypercholesterolemic (HC) rabbits exhibit suppressed compensatory vascular growth after restriction of arterial supply. However, neovascularization is commonly found in atheromas containing inflammatory cells. We used an in vitro model to determine the effects of hypercholesterolemia on angiogenesis in the absence or presence of inflammatory cells. HC rabbit aortic explants (1 mm2) with or without (n=90 each) lesion-forming inflammatory cells were cultured in a collagen matrix with serum-free medium. Explant-derived endothelial cell growth was organized into capillary-like microtubes (CLM) that could be videomicroscopically quantified. CLM growth from lesion-free HC explants was significantly reduced to 13±4% of the value in explants (n=90) from normocholesterolemic (NC, n=15) rabbits (P<.001). In contrast, in lesion-containing HC explants, the matrix was invaded by foam cells, and CLM growth was not inhibited. Immunoassayable basic fibroblast growth factor (bFGF, in pg/mL) in the culture medium was significantly lower in lesion-free HC (<5) than NC explants (11±2, P<.01) or HC explants with lesions (14±3). In addition, CLM growth was reduced in NC explants incubated with oxidized LDL (ox-LDL, 50-100 µg/mL). Exogenous bFGF (10 ng/mL) reversed the inhibitory effects of hypercholesterolemia and ox-LDL, whereas bFGF-neutralizing antibody (10 µg/mL) abolished CLM growth in all groups. In cultured rabbit aortic endothelial cells, ox-LDL reduced DNA synthesis, but this inhibition was reversed by bFGF. We conclude that hypercholesterolemia and ox-LDL inhibit angiogenesis-like endothelial growth because of a suppressed availability of endogenous bFGF. Retained responsiveness to exogenous bFGF suggests that inducing bFGF expression at targeted sites may improve collateral growth in hyperlipidemic arterial disease.
Key Words: angiogenesis • hypercholesterolemia • oxidized LDL • basic fibroblast growth factor
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Introduction |
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Like luminal expansion, angiogenesis can represent compensatory vascular remodeling to help maintain organ perfusion in the presence of insufficient arterial supply. The response has been described in humans1 2 and in experiments with animals.3 4 The mechanisms of compensatory angiogenesis are a focus of research interest, in particular because of potential therapeutic implications.
Recent studies have demonstrated that administration of growth factors such as bFGF and VEGF can stimulate angiogenesis in NC animals.5 6 7 However, whether the release and effector functions of these growth factors remain intact or are impaired in the presence of dyslipidemic cell injury has not been fully evaluated. Previously, we demonstrated that adaptive growth of arteries and microvessels in response to surgical restriction of arterial supply was markedly suppressed in HC rabbits.8 In a more recent study, we showed that atherosclerotic impairment of angiogenesis in human coronary arterial explants can be reversed by exogenous bFGF.9
The present in vitro experiments with tissue explants from rabbits were designed to further characterize the role of bFGF in angiogenesis in a dyslipidemic environment. Neovascularization may occur in atheromatous lesions,10 but such localized angiogenesis is stimulated predominantly by the inflammatory process of lesion formation.11 12 Compensatory vascular growth usually originates in lesion-free areas. We quantified angiogenesis, release of endogenous bFGF, effects of exogenous bFGF, and effects of bFGF-neutralizing antibody in explants with and without atheromatous lesions from HC rabbits. Also, using explants from NC rabbits, we assessed these variables according to exposure or nonexposure to ox-LDL. Oxidatively modified lipoproteins suppress endothelial replication,13 14 15 a phenomenon that could limit vascular growth in vivo.
A rabbit aortic explant culture model was used so that interactions among dyslipidemia, mononuclear inflammatory cells (macrophage foam cells), and bFGF could be assessed in the absence of hemodynamic or systemic humoral factors. The in vitro model permitted us to compare the effects of growth factors and their neutralizing antibodies on organized endothelial growth in normal and atherosclerotic arterial tissue that was uninfluenced by the conventional cell culture conditions necessary to support endothelial cells. By use of an ELISA, explant-derived bFGF in each well was quantified and compared with endothelial response.
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Methods |
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Animals
Male New Zealand White rabbits (2.5 to 3.0 kg) were randomized for 8 weeks to standard (n=15) or 1% cholesterol–supplemented (n=15) rabbit chow (Nutrition Biochem), yielding NC and HC groups, respectively (mean±SE plasma total cholesterol, 1.2±0.3 and 17.5±2.3 mmol/L, respectively; P<.001). The rabbits were killed after 8 weeks with pentobarbital (100 mg/kg body wt IV), and the descending thoracic aorta was rapidly excised.
Preparation of Native and Ox-LDL
Native LDL (d=1.019 to d=1.063 g/mL) from
pooled human plasma anticoagulated with EDTA (1 mg/mL) was isolated by
sequential ultracentrifugation.16 17 To
prepare ox-LDL, native LDL was exposed for 24 hours at 37°C to
cultured RAECs (a gift of Dr Joseph Witztum, University of California,
San Diego) or 5 µmol/L CuSO4 in
PBS.16 17 After incubation, LDL was reisolated by
ultracentrifugation (flotation at d=1.210
g/mL) with 1 mg/mL EDTA. Preparations were dialyzed against
Krebs-Henseleit buffer formulated as previously
described.17 Precautions were taken to prevent endotoxin
contamination during lipoprotein isolation and oxidation, including the
use of pyrogen-free sterile water for all reagents. All glassware was
washed with 1% E-Toxa-Clean in sterile water, rinsed with sterile
water, and baked at 185°C for 5 hours.18 Endotoxin
contamination was monitored with the coagulation Limulus
amebocyte lysate assay using Pyrotell sensitivity (
=0.25
EU/mL) as the standard in serial dilutions (Associates of
Cape Cod, Inc). The endotoxin concentration of the ox-LDL preparations
was <0.1 EU·mL-1·mg-1 protein.
Protein content in LDL preparations was estimated by the method of
Lowry et al with bovine albumin as the standard. Thiobarbituric
acid–reactive substances in LDL preparations were assayed as a measure
of oxidative lipid modification.16 17
Organ Culture
The organ culture technique used in this study was developed
independently9 but has features in common with the aortic
ring culture models reported by Nicosia, Kawasaki, and their
associates.19 20 21 22 After removal of the adventitia under
microscopic visualization, intima-media explants (1
mm2) were cut from the thoracic aorta with the use of a
McIlwain tissue chopper (The Mickle Laboratory Engineering Co Ltd) set
at 1 mm for both dimensions. Rat tail collagen type I solution
(Collaborative Biomedical Products), 10x RPMI 1640 medium (GIBCO
Laboratories), and 240 mmol/L NaHCO3 (8:1:1,
vol/vol/vol) were mixed at 0°C. After the pH was adjusted to 7.4 with
1N NaOH, the reconstituted solution (300 µL per well, Corning 24-well
culture plates) gelled in 10 minutes at 37°C. Single explants placed
endothelial side down in the center of each well were
covered with another 300 µL of collagen mixture, which entrapped the
explants between two layers of collagen matrix. Serum-free RPMI 1640
medium (500 µL per well) containing penicillin (100 IU/mL) and
streptomycin (100 µg/mL) was added. Cultures were incubated at 37°C
in a 95% O2/5% CO2 atmosphere, and the medium
was changed every other day. All explants were cultured for 14 days
unless specified otherwise. After 2 or 3 days, sprouting CLM invaded
the thin layer of collagen substrate and extended away from the edges
in the plane of the fused collagen layers. CLM growth was visualized by
serial video-microscopic imaging with a Nikon inverted microscope and a
Cohu model 5172 video camera. Video images were analyzed by
computer equipped with a Targa M8 video digitizing board and installed
with a JAVA image analysis program (Jandel Scientific). The
lengths of individual microtubes, including their branches, were
determined by delineating their axes on the video screen with an
electronic cursor. The sum of lengths of the axes was computed as an
index of angiogenesis-like response, a procedure that combines criteria
of CLM length and density that have been used previously in the
assessment of CLM outgrowth from rat aortic rings.19 20 21 22
Because CLM arises exclusively from the cut edge of the
explant,9 21 the sum of CLM lengths was initially
normalized for explant circumference. However, the ratios of explant
circumference to explant were, on average, very similar in different
groups. Therefore, unless specified otherwise, in this study the index
is expressed as the sum of CLM lengths per explant.
Histology and Immunochemistry
Collagen containing the explants after selected culture periods
and tissue bordering the explants were fixed in 4%
paraformaldehyde and embedded in paraffin.
Deparaffinized sections were stained with hematoxylin and eosin for
histology or incubated with specific antibodies for immunochemistry. To
identify endothelial, smooth muscle, and rabbit
macrophage cells,23 the sections were incubated
for 60 minutes with antibodies to vWF (1:200, Incstar), 
,
-actin
(HHF35 1:1, Enzo), and RAM11 antigen (1:100, Dako), respectively. An
immunoperoxidase procedure (Vectastain, Vector Labs) performed in
conjunction with species-specific biotinylated secondary antibodies was
used to visualize bound primary antibody. Control stains were performed
by using primary antibodies to irrelevant antigens or omitting the
primary antibody. To visualize the antigen-antibody complex, sections
were incubated with a diaminobenzidine solution, rinsed in distilled
water, and counterstained with hematoxylin.22 To
demonstrate lipid inclusion in macrophages, fresh tissues were
stained with oil red O.
Transmission Electron Microscopy
To evaluate the ultrastructure of cell outgrowth from the
explants, fragments of collagen containing the explants were fixed in
3% glutaraldehyde buffered with 0.1 mol/L PIPES
overnight. Specimens were then rinsed in PIPES buffer, treated with 2%
OsO4 for 1 hour, and embedded in Spurr's resin after
serial dehydration. Serial 1-mm sections stained with toluidine
blue/basic fuchsin were examined by light microscopy. Sections
containing endothelial microtubes were cut into 60-nm
sections, stained with uranyl acetate/lead citrate, and examined in a
JEOL 100C transmission electron microscope.
NC Versus HC Explants
The explants were cut from predetermined sites that corresponded
to lesion-resistant areas (ventral descending thoracic aorta
maximally remote from intercostal artery ostia and designated site 1)
or lesion-prone areas (aortic arch designated site 2; Fig 1
). For each treatment group, 6 explants from each
rabbit were cultured. In NC explants, CLM growth was not different
between sites 1 and 2 (see "Results"). In the absence of serum or
exogenous growth factors, time-dependent CLM growth of NC explants was
compared with that of HC explants from site 1 or 2. In some
experiments, site 1 HC explants were cocultured with site 2 HC explants
to ascertain whether diffusible factors from site 2 explants might
stimulate growth of site 1 explants. We use the term
"lesion-free" to designate exclusively those explants obtained
from lesion-resistant areas (site 1) of HC aortas.
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Treatment With Native and Ox-LDL
Because oxidatively modified LDL exerts inhibitory
effects on the growth of endothelial cells in
culture,14 15 we incubated NC explants with native or
oxidatively modified LDL preparations. LDL preparations were added to
the medium on the first and third days of culture.
Simultaneous experiments with NC explants cultured without
or with graded concentrations (10, 50, or 100 µg/mL) of ox-LDL or
native LDL (100 µg/mL) were conducted. To determine whether the LDL
preparations exerted direct cytotoxic effects, cultured RAECs were
incubated with 100 µg/mL native LDL or 100 µg/mL ox-LDL for 24 or
48 hours. DNA synthesis was evaluated by including in the medium 3
µCi/mL [3H]thymidine (Moravek Biomedicals) during the
last 6 hours of a 24-hour incubation period.24 Cells were
counted with a hemacytometer, and the percentage of dead cells was
determined by trypan blue positivity.
Treatment With Growth Factors and Growth Factor–Neutralizing
Antibodies
To characterize responsiveness to established
angiogenesis-modulating factors, explants were treated every other day
with 10 ng/mL TGF-ß1, 10 ng/mL TNF-, 10 ng/mL VEGF, or 5 to 20
ng/mL bFGF (all from R&D Systems). To determine whether
endogenous bFGF was important for CLM growth, NC and
lesion-containing HC explants were also treated every other day with 10
µg/mL bFGF-neutralizing antibody (goat IgG, R&D Systems). For
comparison, some explants were treated with 10 µg/mL
TGF-ß1–neutralizing antibody (R&D Systems) or 10 µg/mL nonimmune
goat IgG (Sigma Chemical Co). To evaluate the reversibility of
ox-LDL–induced effects on DNA synthesis, cultured RAECs with or
without exposure to ox-LDL were treated with 2 ng/mL bFGF.
Measurement of bFGF Concentrations
Minute bFGF concentrations can be accurately detected by
sensitive ELISA in the culture medium during the early hours of in
vitro angiogenesis.25 To determine whether the levels of
endogenously released bFGF were related to CLM growth, bFGF
concentrations in the culture medium were measured by sandwich ELISA
with the use of the human FGF basic Quantikine kit (R&D Systems,
catalog No. DFB00). bFGF standards (provided in the kit) and medium
samples were incubated in wells of the microtiter plate coated with a
monoclonal antibody specific for bFGF at room temperature for 2 hours.
After the plates were washed, a conjugate containing a polyclonal
antibody against bFGF was added and the complex was incubated for
another 2 hours. After the substrate solution was added, the optical
density of each well was assayed in a spectrophotometer with the
wavelength set to 450 nm. bFGF concentrations of the medium samples
were determined with the use of standard curves. bFGF concentrations in
the plasma from NC or HC rabbits were also measured. Although the
manufacturer indicated that the minimum detectable concentration was 1
pg/mL, values <5 pg/mL determined with the standard curves, as
recommended, lacked precision and therefore have been reported as <5
pg/mL.
Statistical Analysis
Differences between group means were compared using either
paired t tests for single comparisons or modified
t statistics (Bonferroni test) for multiple comparisons. An
ANOVA followed by Scheffé's test for significance was used to
compare time-dependent CLM growth between groups. A value of
P<.05 was considered significant. Values are reported as
mean±SE.
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Results |
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CLM Emerging From Normal Aortic Endothelium
As shown for human coronary and rat aortic ring cultures,9 19 20 21 22 endothelial cells formed microtubes that sprouted from explant edges and underwent progressive elongation (Fig 2A
). The growing microtubes had a
capillary-like ultrastructure, with single layers of
endothelial cells surrounding the lumen (mean luminal
diameter, 10±4 mm) containing no collagen (Fig 2B). The cell
lining of the lumen stained with antibody against vWF, an
endothelial marker; this staining did not occur with
omission of the primary antibody (Fig 2C and 2D). There were no
vWF-positive cells that were not associated with the microtubes. In
contrast, isolated spindle-shape cells emerging from explant edges
stained for muscle actin (HHF35 antibody) (data not shown). These cells
probably represented smooth muscle cells or myofibroblasts
and were not associated with the microtubes.21 Although
explant-derived CLM lacked well developed basement membranes and
pericytes, several features of CLM growth resemble angiogenesis in
vivo.3 26 The angiogenesis-like features include
sprouting, progressive elongation, and penetration of the CLM through
the interstitium-like collagen substrate.
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Microvascular structures originating in the planar explants were
located within a thin layer of collage, which resulted in a
predominantly two-dimensional growth distribution. Serial video
microscopic images showed that after an initial lag phase of 2 days,
during which time "primordial" endothelial buds
developed at the edges of the explants, there was an elongation of
microtubes that progressed nearly linearly for 3 to 5 days, after which
growth halted. After 10 days, the microtubes began to degenerate (Fig 3). In pilot studies with NC explants, the day-7 CLM
growth index for lesion-resistant site 1 explants
(2.45±0.28 µm per explant, n=24; 6x4) was not different from
that for lesion-prone site 2 explants (2.37±0.31 µm per
explant, n=24). Explants from site 1 were used to represent the
NC explants, and the mean 7-day angiogenesis index was 2.40±0.21
µm per explant (n=90, 6x15). Expressed with respect to explant
circumference, the index was 0.61±0.07 µm/mm.
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Effect of Dietary
Hypercholesterolemia on CLM Growth
Light microscopic examination of tissue surrounding
explants revealed no intimal thickening and a complete absence of
mononuclear leukocytes or lipid-laden macrophages at HC
lesion-resistant site 1 (Fig 1
). Compared with NC explant
controls, these explants invariably exhibited very poor growth, with a
day-7 index of 0.31±0.12 µm per explant (0.07±0.03
µm/mm, P<.001), or 13±4% of controls. The index,
expressed with respect to the circumference of individual explants, was
(P<.001). The poorly developed CLM degenerated quickly
after day 7 and disappeared almost completely after 14 days (Fig 3).
There were no foam cells surrounding lesion-free HC explants (Fig 4A).
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Tissue surrounding HC lesion-prone site 2 explants contained abundant
subendothelial macrophage foam cells that
stained positively with RAM11 antibody and oil red O. These cells
invaded the matrix, and some of them were later observed in areas
distant from the explant edges (Figs 1 and 5).
Intraintimal neovascularization could not be demonstrated during
examination for vWF-positive cells (data not shown). In contrast to
lesion-free site 1 HC explants, lesion-containing site 2 explants
showed no growth inhibition (day-7 index, 3.05±0.44 µm per
explant, or 0.73±0.13 µm/mm, P>.1 compared with NC
controls; Figs 3 and 4B). Another distinguishing feature was the
invariable association of RAM11-positive macrophage foam
cells that were apposed to the proximal and distal segments of the CLM
(Figs 4B and 5). Electron photomicrographs revealed intimate contact
between the abluminal surface of the endothelial cells
and the macrophages (Fig 6). However, the
presence of macrophages did not appear to be associated with
changes in the ultrastructure of the CLM (Figs 2 and 6). In contrast to
the CLM growth pattern in NC explants, the CLM continued to grow in
these macrophage-rich explants until day 10 and showed only a
slight decline after 14 days (Fig 3).
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), two exhibiting clear vesicles (foam cell), surround the
endothelial microtube. C indicates collagen fibers; EX,
explant edge; and J, intercellular junction.
Coculture Experiments
To demonstrate that lesion-containing explants released
proangiogenic factors that were missing from lesion-free explants,
coculture experiments were performed. Lesion-free HC explants exposed
to cocultured lesion-containing explants from the same aorta exhibited
a threefold increase in CLM growth (1.20±0.26, n=24,
P<.05), with growth most marked in the vicinity of the
lesion explants.
Effects of Ox-LDL
Native LDL (100 µg LDL protein per milliliter) prepared without
antioxidants (other than EDTA) exerted a nonsignificant effect on CLM
growth in NC explants. In contrast, ox-LDL dose-dependently (10 to 100
µg/mL) inhibited CLM growth (Table 1). In cultured
RAECs, 100 µg/mL ox-LDL decreased thymidine incorporation into DNA at
24 hours without increasing the percentage of dead cells, whereas 100
µg/mL native LDL had no effect. The cell counts were not different
from those for untreated controls (Table 2).
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Effect of Exogenous bFGF on CLM Growth and DNA Synthesis
In NC explants treated with 10 ng/mL TGF-ß1 or 10 ng/mL TNF-,
CLM growth was reduced to <50%; CLM growth in explants treated with
10 ng/mL VEGF was not different from that of controls (Table 3). In contrast, bFGF dose-dependently (5 to 20 ng/mL)
increased CLM growth in NC explants (Table 3 and Fig 7).
Moreover, microtube growth was sustained for 14 days, after which
degradation became noticeable (Fig 7). bFGF partially corrected growth
suppression in lesion-free HC explants and NC explants incubated with
ox-LDL (Table 4).
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In cultured RAECs not exposed to LDL preparations, bFGF increased DNA
synthesis twofold and cell count by 20%. In cells treated with ox-LDL,
bFGF corrected the replication deficit and produced increases in DNA
synthesis that exceeded those observed in untreated cells (Table 2).
Role of Endogenous bFGF on CLM Growth
Treatment with bFGF-neutralizing antibody at the beginning of the
culture nearly abolished CLM growth in NC explants. In
lesion-containing HC explants, bFGF-neutralizing antibody did not block
the emigration of foam cells but did abolish CLM growth to the same
extent as in the NC group (Figs 4C and 8). When
bFGF-neutralizing antibody was given on day 10 to HC explants with
lesions, the well developed CLM underwent rapid fragmentation and
degeneration within 48 hours. This change suggested that the growth
factor contributed not only to the growth but also to the preservation
of the CLM. In NC explants or HC explants containing foam cell lesions,
treatment with 10 ng/mL TGF-ß1–neutralizing antibody or 10 ng/mL
nonimmune IgG did not affect CLM growth (Figs 4D and 8).
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Correlation Between Endogenously Released bFGF and
CLM Growth
As shown in Table 5, bFGF concentration (in
picograms per milliliter) in the media of day-5 cultures of NC explants
(11±2) was similar to that of HC explants (14±3) containing lesions.
In contrast, bFGF concentration in the media of lesion-free HC cultures
was significantly lower (<5) than that of the other groups. Treatment
of NC explants or lesion-containing HC explants with bFGF-neutralizing
antibody significantly reduced measurable bFGF. Treatment with
TGF-ß1–neutralizing antibody or nonimmune IgG had no effect. The
plasma bFGF concentration of HC rabbits (6±1, n=6, P<.05)
was lower than that of NC rabbits (10±2).
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Discussion |
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In this study, we demonstrated that rabbit aortic explants in organ culture gave rise to an organized, capillary-like growth of endothelial cells. In HC rabbits, lesion-free explants containing no immunostainable macrophages exhibited markedly suppressed CLM growth. When explants were occupied by lesion-forming inflammatory cells, however, endothelial microtubes were surrounded by emigrating macrophages, and organized endothelial growth was not suppressed or even enhanced. Ample growth in the NC group and in the lesion-containing HC group was sensitive to the inhibitory effects of a bFGF-neutralizing antibody. Exogenous bFGF, on the other hand, substantially stimulated growth in the lesion-free HC group. In combination, these results suggest that suppressed growth in the lesion-free HC group reflected a reduced availability or possibly production of bFGF and that the enhanced growth in the presence of emigrating macrophages was mediated by a bFGF-dependent mechanism.
Restriction of the arterial blood supply determines an increased flow through residual arterial pathways. Adult arteries chronically exposed to high flow are known to undergo adaptive growth and remodeling with modulation in bFGF expression.27 28 Because hypercholesterolemia may impair flow-dependent regulatory mechanisms,29 30 it is possible that atherosclerotic arteries become insensitive to the growth-stimulatory effects of flow loads. In this study, we demonstrated dyslipidemic effects on angiogenesis in the absence of flow alterations.
Although new capillaries usually arise from parent capillaries,31 32 endothelial cells derived from a large artery (eg, human coronary or rat aorta) are capable of forming CLM in a serum-free organ culture system.9 19 20 21 22 In our model, the matrix provided a three-dimensional, interstitium-like environment that promoted organized endothelial growth. CLM originating in the edges of the explant were located in the plane of the endothelial monolayer of the explant. The thinness of the collagen coating the wells and possibly the sequential layering of two collagen coats facilitated the predominantly planar growth of the microtubes. Serial observations showed that elongation of the CLM occurred slowly over a period of days. As in other in vitro angiogenesis models,31 32 microtubes growing from the explants lacked the organization of mature capillaries. However, the formation of sprouts and subsequent progressive elongation and branching are features in common with the early stages of capillary angiogenesis in vivo.3 4 26 The slow, progressive pattern in the absence of endothelial cells unassociated with microtube assembly differs from the rapid (within 1 to 2 hours) tube-forming rearrangements of endothelial cells plated on a Matrigel matrix. Microtube formation under the latter conditions occurs with little or no endothelial cell proliferation.33 In contrast, in rat aortic ring cultures, endothelial cells forming capillaries incorporate BrdU into their nuclei,20 a response suggesting the importance of cell replication. The formation of microtubes in our experiments indicates that macrovascular aortic endothelium can undergo phenotypic changes promoting microvessel-like cellular assembly.21 Elongation of microtubes has been suggested to reproduce in vitro the processes of angiogenesis in vivo, including proteolytic basement membrane breakdown, migration, proliferation, and tube formation with intraluminal collagenolysis.26 34 The major strength of the model is that the organ culture system is not dependent on exogenous mitogens (eg, serum, growth factors, or cytokines). Therefore, the model appears suitable for the detection of missing factors (or excess inhibitors) regulating cell growth.
For quantification of microtube growth, we used a method similar to that employed in studies of rat aortic ring explants.19 20 21 22 The spatial distribution of microtubes in a single plane facilitated their morphometric quantification. Our conclusions are not critically dependent on the precision of the quantitative morphometric method, because semiquantitative assessment would have sufficed to demonstrate the striking effect of hypercholesterolemia in the absence of inflammatory cells (<15% of control). Previous experiments in rabbits suggested that compensatory growth of large and small arteries induced by surgical restriction of the arterial blood supply is markedly suppressed.8 Results of the present study indicate that an atherogenic dyslipidemia influences intrinsic growth properties of artery cells that do not depend on flow-sensing mechanisms or systemic humoral effects. The observed growth impairment cannot therefore be ascribed to a defective shear stress–dependent gene activation mechanism.
Of interest is the observation that ox-LDL inhibited CLM growth in explants from NC rabbits. This finding suggests that the uptake or intracellular generation of ox-LDL by endothelial cells in vivo might play a role in impairing vascular growth.35 36 At 24 hours, ox-LDL reduced premitotic DNA synthesis in cultured RAECs in the absence of a change in cell number. These results suggest that the ox-LDL–induced inhibition of CLM growth did not reflect lethal cytotoxic effects. Reversal of inhibition in response to bFGF or explants in coculture supports the view that hypercholesterolemia and ox-LDL acted without causing irreversible cell injury. We have previously reported that LPC, a phospholipid metabolite accumulating in atherosclerotic arterial walls and ox-LDL, inhibits DNA synthesis of endothelial cells.9 Recently, Murugesan and Fox37 demonstrated that LPC exerts antimigratory effects on endothelial cells. Because migration of endothelial cells plays a pivotal role in angiogenesis, LPC is one factor that might contribute to inhibited angiogenesis mediated by ox-LDL.
Effects of TGF-ß1 and TNF- on endothelial cell
growth are complex and appear to depend on growth factor concentration
and substrate conditions. Although these agents may inhibit
endothelial cell growth in two-dimensional cultures,
low concentrations may actually stimulate tube formation in
three-dimensional matrices.12 In our model, both growth
factors in high concentrations exerted inhibitory effects
on CLM growth. VEGF did not increase or decrease CLM growth in this
model. In contrast, bFGF, an angiogenic factor both in vivo and in
vitro, increased CLM growth in NC explants. Growth suppression in these
explants by bFGF-neutralizing antibody indicated the importance of
endogenous bFGF in the angiogenic response. The importance
of endogenous bFGF in angiogenesis has also been
demonstrated in cultures of aortic rings from NC rats.38
In preliminary studies, we confirmed that bFGF can induce expression of
its own gene,39 40 and we found that ox-LDL decreased bFGF
gene expression in vascular endothelial
cells.41 Recent experiments in our laboratory indicate
that steady-state bFGF mRNA levels can be dose-dependently reduced by
ox-LDL in cultured endothelial cells.41
These findings suggest that decreased production of bFGF is
responsible for suppressed angiogenesis in the presence of
dyslipidemia.
When explants contained inflammatory cell–rich lesions (ie, fatty streaks), CLM growth was activated and the growth of CLM was invariably accompanied by macrophage foam cells in close apposition to the outer surface of the microtubes. This finding suggests that endothelial cells exposed to a dyslipidemic environment, although possessing an intrinsically suppressed capacity for replication and organized growth, can still respond to external or paracrine angiogenic stimuli. It also indicates that neovascularization of atheromatous lesions reflects predominantly a local angiogenesis stimulated by the inflammatory processes of lesion formation.10 11 Because atheromatous neovascularization is a local phenomenon, its presence does not imply that adaptive growth of mature vessels is preserved in the presence of hypercholesterolemia.
Numerous angiogenic factors have been demonstrated to be released from macrophages undergoing inflammatory activation.12 The close approximation between macrophages and microtube-forming endothelial cells is likely to facilitate paracrine interactions between these two cell types. Although contributions by other growth factors or cytokines such as VEGF,7 42 acidic FGF,11 43 or interleukin-844 expressed by macrophages cannot be ruled out, the degree of suppression of CLM growth by bFGF-neutralizing antibodies suggests a strong dependence on bFGF. The specificity of the bFGF contribution was confirmed by the absence of inhibitory effects of TGF-ß1–neutralizing antibody and nonimmune IgG.
bFGF, a ubiquitous growth factor expressed by many cells including endothelial cells and macrophages,12 43 exerts its multiple effects by means of autocrine and paracrine pathways.39 45 Although it lacks a typical hydrophobic signal-peptide sequence, bFGF synthesized by endothelial cells has been demonstrated to be deposited in the subendothelial extracellular matrix.46 This indicates that bFGF can be secreted by a nonclassic pathway that is apparently independent of the endoplasmic reticulum–Golgi complex.47 With recent improvements in immunoassay techniques, elevated bFGF levels have been detected during the early hours of in vitro angiogenesis.25 In this study, bFGF was detected in the serum-free media of explant cultures. The concentrations of secreted bFGF in the media correlated well with the degrees of CLM growth. In the poorly growing lesion-free HC group, bFGF concentration was also extremely low. Increased bFGF release from lesion-containing HC explants promoted CLM growth. Because activated macrophages are known to secrete numerous growth factors, including bFGF,12 one plausible explanation is that lesion-derived macrophages were a source of bFGF that could correct the defective growth of endothelial cells exposed to hypercholesterolemia. The paracrine effect of bFGF may contribute to localized neovascularization of atheromas. Although bFGF may be secreted by inflammatory and activated endothelial cells in vivo, it is interesting to note that plasma bFGF concentrations were lower in HC than in NC rabbits. Whether these lower concentrations play a role in limiting compensatory macrovascular and microvascular growth after surgical restriction of the arterial blood supply in vivo is uncertain, however. Elucidation of underlying biochemical or genetic mechanisms accounting for the reduced availability of bFGF will require detailed investigation. In addition, it will be important to determine in monoculture systems the comparative effects of ox-LDL on bFGF expression by endothelial cells and macrophages. Maintained responsiveness of HC explants with suppressed growth to exogenous bFGF suggests that receptor and transduction functions for the factor are partly preserved, but effector pathways of bFGF in HC states will require further characterization.
In conclusion, endothelial cells of a large artery exhibit a bFGF-dependent capillary-like growth in a three-dimensional, interstitium-like matrix. Exposure of the cells to elevated cholesterol concentrations in vivo or to ox-LDL in vitro produces growth impairment that can be partly reversed by exogenous bFGF. Stimulation of the endogenous production of angiogenic mitogens or administration of orally active factor analogues is an intriguing option for the treatment of hyperlipidemic occlusive arterial disease.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by grants HL-40884 and HL-36894 from the National Institutes of Health, Bethesda, Md; grant and award 38520 from The Methodist Hospital Foundation, Houston, Tex; and Grant-in-Aid 95G-239 from the American Heart Association, Texas Affiliate, Austin, Tex. The authors are grateful to Suzanne Simpson for her excellent editorial assistance.
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Footnotes |
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Presented in part in abstract form at the 41st American College of Cardiology Annual Scientific Sessions, April 12-16, 1992, Dallas, Tex; the 65th Scientific Sessions of the American Heart Association, November 16-19, 1992, New Orleans, La; and the 10th International Symposium on Atherosclerosis of the International Atherosclerosis Society, October 9-14, 1994, Montreal, Quebec, Canada.
Received May 31, 1996; accepted November 11, 1996.
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