Atherosclerosis and Lipoproteins |
From the Institute of Anatomic Pathology, Tor Vergata University of Rome, Rome, Italy.
Correspondence to Prof Augusto Orlandi, Institute of Anatomic Pathology, Department of Biopathology, Tor Vergata University of Rome, Via della Ricerca Scientifica, 00133, Rome, Italy. E-mail orlandi{at}uniroma2.it
| Abstract |
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Key Words: atherosclerosis aging smooth muscle cells glycosaminoglycans endothelial nitric oxide synthase
| Introduction |
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| Methods |
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Plasma Analysis
Blood samples were drawn from the marginal vein of fasted
rabbits (12 hours), with the addition of EDTA (Merck). Plasma was
separated by centrifugation at 3000 rpm for 20 minutes,
and an aliquot was used for lipoprotein separation, as we previously
reported.27 Briefly, VLDL (density [d] 1.006
g/mL), IDL (d 1.006 to 1.019 g/mL), LDL (d 1.019
to 1.065 g/mL), and HDL (d 1.065 to 1.21 g/mL) were
separated by ultracentrifugation in a discontinuous
salt gradient by an IEC-60 ultracentrifuge (International
Equipment Co) at 41 000 rpm for 24 hours at 14°C. Fractions were
collected by using a tube slicer (Beckman Inc) and stored at -20°C.
Plasma lipemic pattern and lipoprotein composition were determined by
enzymatic methods27 28 with the use of an
Auto-analyser Cobas Mira S (Roche).
Morphometric Studies
To evaluate the size and the extent of lesions,15
the intimal volume and surface area relative to the
arterial wall were calculated on Movats
pentachromestained sections of entire rolled aortas of
perfused rabbits by using a Quantimet 920 image analyser (Cambridge
Instruments) connected to a Polyvar microscope (Reichert Jung) by a
Hamamatzu HC3077 camera. To evaluate the possible differences in the
distribution,17 a macroscopic mapping of lesions of the
thoracic aorta was manually performed on each double-sized photograph
by 2 different researchers using a 30x30-cm grid with 2x2-mm squares
and a line spacing of 0.11 mm. Their intervariability was l<5%.
Lesions were arbitrarily classified as adjacent or distant (situated at
2 mm from the ostia) from branches. The prevalent spatial
relation with the ostia (ie, upstream, lateral, and downstream) was
also recorded.
Differences in phenotype, proliferation, and apoptosis were quantified by calculating the percentage of positive cells or their number per unit of area. The number of fields required to obtain a significant difference was calculated according to a stereological formula.29 Intimal cellularity was obtained by calculating the number of cells per unit area at x200 magnification. A minimum of 20 random fields and 5 serial sections were evaluated by 2 different researchers, with intervariability <5%.
A semiquantitative evaluation of glycosaminoglycans (GAGs) of the extracellular matrix was performed by using Alcian blue (8GX, Sigma; pH 2.7)stained paraffin sections of rolled perfused aortas according to the method of Wight et al,30 with modifications.31 Specificity of Alcian blue staining was controlled on serial sections by digestion with 0.1% testicular hyaluronidase (Sigma) in sodium acetate buffer, pH 5.4, and 0.5 U/mL chondroitinase ABC (Sigma) in Tris buffer before staining. Alcianophilia was estimated at x200 magnification by 2 different researchers who used a grading system in arbitrary units as follows: 0, absent staining; 0.5, equivocal staining; 1, faint staining; 2, moderate staining; and 3, intense staining. The interobserver reproducibility was >95%. For each animal, the ratio of the resulting score with the total number of fields analyzed was calculated.
Ultrastructural Study
For transmission electron microscopy, small aortic samples
selected from the arch and the thoracic tract of perfused aortas were
postfixed in 1% OsO4 for 2 hours and dehydrated
through an alcohol series and propylene oxide before they were embedded
in Epon 812. Thin sections were stained with toluidine blue to verify
the absence or presence of lesions. Ultrathin sections were cut by an
8800 Ultramicrotome III (LKB), counterstained with uranyl acetate and
lead citrate, and studied with a Philips 301 electron microscope. To
verify the presence of GAGs, small samples after fixation were
transferred to wash buffer (0.15 mol/L sodium cacodylate) containing
ruthenium red (0.75 mg/mL) for 16 hours. After postfixation in 1%
OsO4 plus ruthenium red (0.75 mg/mL) for 2 hours
and en bloc staining in 10% aqueous uranyl acetate for 1 hour,
dehydration and embedding were performed as reported above.
For scanning electron microscopy (SEM), arterial fragments were dehydrated in ethanol and acetone series and dried in an E3100 critical point drier (Polaron Equipment Limited) with CO2 transition fluid. Specimens were mounted on aluminum stubs with silver print and coated with a 20-nm gold layer in an E500-PS3 sputter-coater (Polaron). Photographs were made by using a SCAN100 scanning electron microscope (Cambridge Instruments) at 10 kV. The number of monocytes attached per 1 mm2 of endothelial surface was counted on photographs at x400 magnification of at least 12 randomly selected areas for each rabbit, excluding sites at the edge of specimens. In hyperlipemic animals, monocytes adhering to endothelium overlying (or not) atherosclerotic lesions were counted separately, and in normocholesterolemic rabbits, monocytes in areas far or adjacent (<2 mm) to the branches were counted separately.
Immunohistochemistry and Terminal
Deoxynucleotidyl TransferaseMediated dUTP-Biotin
Nick End-Labeling
Serial sections of rolled perfused aortas were deparaffinized,
rehydrated, treated in sequence with 3%
H2O2 and normal rabbit
serum, and incubated with RAM 11, a monoclonal antibody to rabbit
macrophages,32 or anti
-smooth muscle actin
(
-actin).33 Lymphocytes were quantified on
ethanol-fixed cryostatic sections from aortic rings (Table 1
) by an overnight reaction with a mouse
anti-rabbit CD5 (clone KENB-5, Serotec), which recognizes rabbit T
lymphocytes.34 Previous studies on serial sections that
reacted with
-actin and RAM 11 demonstrated that the percentage of
positive cells did not significantly differ from paraffin section
values (data not shown). The expression of endothelial
nitric oxide (NO) synthase (eNOS) was evaluated on methanol-fixed
cryostatic sections by using a monoclonal antibody (Zymed Laboratories
Inc). Preliminary studies demonstrated the specific positive eNOS
reaction of rabbit endothelial cells and the negativity
of SMCs and fibroblasts in various organs. Intimal proliferating cells
were evaluated by incubating aortic and control gut sections with an
anti-BrdU monoclonal antibody (Ylem), as previously
reported.26 All immunostainings were
performed at room temperature. Biotinylated rat-adsorbed anti-mouse IgG
(Vector Laboratories Inc), StreptABC-POD-complex (Ylem), and
diaminobenzidine (Sigma) were used as secondary antibody, revelation
complex, and final chromogen, respectively.
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To indicate apoptosis, rehydrated sections were stripped from proteins by incubation with 300 U/mL proteinase K (Sigma) for 15 minutes at 37°C and endogenous peroxidase blocked with 0.1% H2O2 in methanol (20 minutes) at room temperature. Apoptotic nuclei were revealed by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end-labeling (TUNEL) according to the method of Gavrieli et al,35 with positive controls.
DNA Isolation and Electrophoresis
For genomic DNA isolation, tissue was finely minced in liquid
nitrogen, suspended in 2 mL of digestion buffer (70 mmol/L NaCl,
10 mmol/L Tris-HCl, pH 8.0, 25 mmol/L EDTA, and 1% SDS) with
0.2 mg/mL proteinase K (Sigma) for 16 hours at 50°C. After
inactivation of proteinase K by boiling, samples were incubated with
DNase-free RNase from bovine pancreas (100 µg/mL, Sigma) for 1 hour
at 37°C and extracted twice by phenol/chloroform. DNA was
ethanol-precipitated with 0.3 mol/L sodium acetate (pH 5.0), pelleted
by centrifugation, washed in 70% ethanol, air-dried,
resuspended in Tris-HCl plus EDTA buffer (pH 8.0), and quantified at
260 nm by using an Ultrospec 3000 spectrophotometer (Pharmacia
Biotechnology). DNA extraction and quantification were checked by
electrophoresis on 1% agarose gel containing 1 µg/mL ethidium
bromide under UV light.
Ligation-Mediated PCR
To better identify and quantify DNA fragmentation associated
with apoptosis, we used a ligation-mediated polymerase chain
reaction (PCR) of blunt DNA ends, according to Staley et
al,36 with modifications. Genomic DNA (1 µg) was ligated
to 0.1 nmol each of 24-bp (5'-AGCACTC1'CGAGCCTCTCACCGCA-3') and 12-bp
(5'-TGCGGTGAGAGG-3') unphosphorylated
oligonucleotides. Optimal concentration of
oligonucleotide linker has been previously checked by
serial dilution and by using genomic DNA from AH aortas that
demonstrated the highest percentage of apoptotic nuclei by
TUNEL. Oligonucleotides were annealed by heating to
55°C for 10 minutes. T4 DNA ligase (3 U, Boehringer-Mannheim)
was added, and ligation was performed at 16°C for 16 hours. Reactions
were diluted with Tris-HCl plus EDTA buffer to a final concentration of
5 ng/µL. Ligated DNA (150 ng) was used in the PCR assay (100 µL
volume) containing 124 pmol of the 24-bp linker primer, 67 mmol/L
Tris-HCl, pH 8.8, 3 mmol/L MgCl2, 16
mmol/L
(NH4)2SO4,
10 mmol/L ß-mercaptoethanol, 100 µg/mL BSA, and 320
µmol/L dNTPs (GIBCO). After heating to 72°C for 3 minutes, Taq
polymerase (5 U, GIBCO) was added and incubated at 72°C for an
additional 5 minutes. Samples were amplified by 25 PCR cycles (Perkin
Elmer CETUS) of 1 minute at 94°C and 3 minutes at 72°C. PCR
products (15 µL) were analyzed by 1.2% electrophoretic
agarose gels, equilibrated in 45 mmol/L Tris borate and 1
mmol/L EDTA buffer (pH 8.0), and stained by ethidium bromide (1
µg/mL). To quantify the nucleosomal ladder, gels were photographed,
and negative films were scanned by use of a DuoScan scanner (Agfa)
connected to a Pentium computer. Integrated optical density values of
each lane on the same gel were compared and calculated by using Gel-Pro
Analyze software (Media Cybernetics). PCR and electrophoretic
experiences were repeated in duplicate.
Statistical Analysis
Statistical analysis was performed by use of the SPSS
program (Statistical Package for the Social Sciences, 4th ed, MJ
Norusis/SPSS Inc). For each parameter, the mean, standard
error of the mean, and range were calculated. Differences were
evaluated by t tests and nonparametric
Mann-Whitney tests; values of P<0.05 were considered
statistically significant.
| Results |
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As reported in Table 1
, plasma cholesterol and
lipoprotein levels in the AH and YH groups were similar and greater
than respective normolipemic control levels. No significant differences
were observed when plasma cholesterol and lipoprotein
pattern of the AC and YC groups were compared.
Morphological and Morphometric Findings
Macroscopically, aortic lesions in AH (Figure 1A
) and YH (Figure 1C
) rabbits
appeared as small faint blue or whitish streaks and spots, mainly in
the arch and thoracic tracts. No relevant differences were observed
when AH and YH lesions were compared by Evans blue staining. AH lesions
appeared frequently (58.8±7.2%) distant from arterial
ostia (Figure 1A
); when adjacent, they did not demonstrate any
predilection for upstream, lateral, or downstream margins of the
branches. Instead, 62.3±5.2% of YH lesions were near
arterial branches, generally downstream from the ostia
(Figure 1C
). The difference in the distribution of AH and YH
lesions was statistically significant (P<0.05).
Morphometric analysis demonstrated that the relative surface
area of AH lesions was greater than that of YH lesions
(P<0.02, Figure 2B
),
whereas the intimal relative volume (Figure 2A
) and the
cellularity per millimeter squared (Figure 2C
) did not differ.
No lesions were macroscopically detected either in AC (Figure 1E
) or YC aortas.
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Microscopically, in YH lesions, an endothelial layer
with an irregular profile covered variable accumulations of large
rounded foam cells (FCs) interspindled with rare elongated cells and
scarce extracellular matrix (Figure 3A
).
Very early lesions by a few FCs bulging from the
endothelial surface could also be detected. In AH
lesions, the endothelial profile appeared more regular
than that in YH lesions. Groups or single rounded FCs were mixed with
abundant, sometimes foamy, small elongated cells and a discrete amount
of extracellular matrix, in the absence of extracellular
lipidic-necrotic accumulations (Figure 3B
). These AH lesions
resembled mature fatty streaks or human type II atherosclerotic
lesions.37 Moreover, Movats pentachrome
stain demonstrated the presence of brilliant red
subendothelial accumulations of plasma-like substance
in 80% of AH rabbits and in 46.9% of the observed lesions of AH
rabbits (Figure 3C
). Very small areas of insudate were detected
in only 3.1% of lesions in 1 YH rabbit.
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In the AC group, a diffuse intimal thickening (Figure 3G
) was
observed in the proximal aortic segments of 62.5% of rabbits, without
any predilection for areas adjacent to arterial ostia. The
intimal thickening appeared to be composed of rounded or elongated
cells associated with extracellular matrix, including small elastic
lamellae; no brilliant red areas were observed by Movats
pentachrome stain. In YC rabbits, the tunica intima resulted
from endothelial cells separated from the inner elastic
lamina by a scarce subendothelial space. Two YC rabbits
showed rare accumulations of 1 or 2 subendothelial
cells.
As reported in Table 2
, morphometric
analysis demonstrated that the amount of alcianophilic GAGs of
intimal lesions, such as those underlying the tunica media, was greater
in AH rabbits than in YH rabbits (P<0.01, Figure 1B
and 1D
). In AC rabbits, intimal alcianophilia was marked and more
prevalent than in the underlying tunica media (P<0.02,
Figure 1F
). In YC intima, alcianophilia was practically absent;
in the media, it was scarce and less prevalent than in AH intima
(P<0.02, Figure 1I
).
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Ultrastructural Study
Investigation by transmission electron microscopy confirmed in AH
and YH lesions the variable presence of elongated myocytes and
rounded macrophages, respectively.19 In addition,
rare cells showed a scanty cytoplasm, a few organelles, and a round
nucleus with clumped chromatin, resembling mature
lymphocytes.26 In large AH lesions, semithin and thin
sections sometimes revealed elongated or foamy SMCs surrounded by
fibrous extracellular matrixcovered clusters of rounded macrophagic
FCs, resembling an early fibrous cap (Figure 3D
). No deep
accumulations of extracellular lipidic-necrotic material were observed.
At higher magnification, subendothelial rounded
macrophagic FCs were observed "floating" into AH insudate
(Figure 4A
). In deep portions of
AH fatty streaks underlying the insudate, SMCs were prevalent and
sometimes appeared foamy (Figure 4C
). However, YH lesions showed
a very irregular profile and a marked thinning of
endothelial cytoplasm over the bulging FCs surrounded
by scarce extracellular matrix and rare SMCs (Figure 4B
). We
observed abundant irregularly shaped granules, sometimes showing
filamentous projections, inside the extracellular matrix of AH
lesions. Ruthenium red staining confirmed granules corresponding to
large (20- to 50-nm diameter) and small (10- to 20-nm diameter) GAGs.
GAGs were also present inside the insudate, decorating blocks of
amorphous material (Figure 4D
).
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In AC aortas, intimal thickening resulted from the accumulation of
rounded SMCs, with large amounts of rough endoplasmic reticulum and
cytoplasmic organelles. Extracellular matrix was composed of small
elastic fibers or blocks associated with collagen microfibrils and
ground substance. Ruthenium red staining confirmed that GAGs were
scarce in the subendothelial space of YC aortas (Figure 5A
) and abundant in the extracellular
matrix of AC intimal thickening (Figure 5B
).
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SEM showed the irregular endothelial surface of AH and
YH lesions, with enlarged endothelial intercellular
junctions compared with the adjacent normal tunica intima and single
raised cells that were somehow more evident in YH than AH lesions
(Figure 6
). In YC rabbits,
endothelial cells with a regular, smooth, and intact
profile covered the luminal surface. In AC aortas, the
endothelial cell profile appeared somehow irregular and
prominent. Quantification on SEM photographs (Table 3
) demonstrated an increased number of
monocytes with microvilli attached to the aortic
endothelial surface in hyperlipemic animals compared
with respective normolipemic control animals. As reported in Table 3
, although the overall number of adherent monocytes on lesions
did not differ, those adhering on nonlesioned areas were more numerous
in the AH than in the YH group (P<0.05, Figure 6
).
Monocytes adhering on AH lesions were sometimes grouped, whereas in YH
lesions, they appeared almost single. In AC rabbits, the total number
of adherent monocytes was greater than that in YC rabbits
(P<0.01). This difference was more evident in
endothelial areas far from the branches.
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Immunohistochemistry
Immunohistochemical study on paraffin sections demonstrated a
higher percentage of
-actinpositive cells in AH than in YH lesions
(P<0.02, Figure 2D
). In the latter, RAM 11positive
cells were prevalent (P<0.02, Figure 2E
). In the YH
group,
-actinpositive cells tended to be more frequent in large
than in small lesions, whereas the opposite was true in AH lesions
(Figure 3E
and 3F
). Immunohistochemical study on cryostatic
sections revealed the presence of limited CD-5positive T-lymphocyte
infiltrates in AH and YH lesions. The percentage of CD-5positive
cells was higher in AH than in YH lesions (P<0.02, Figure 2F
). This difference was more marked considering the number of
CD-5positive cells per millimeter squared (19.6±4.9 versus 3.4±1.2,
P<0.01). In AC rabbits, intimal cells appeared to be
-actin positive.
Immunohistochemical evaluation of eNOS demonstrated a faint positivity
of endothelial cells covering AH and YH lesions, such
as in those of YC areas adjacent to branches, and diffuse positivity in
AC endothelium. In contrast to AC cells (Figure 1G
), YC endothelial cells far from the aortic
ostia showed marked positivity (Figure 1H
). Control
-actin
immunoreactivity of underlying medial SMCs on serial sections was
similar (data not shown).
Proliferation and Apoptosis
Anti-BrdU immunostaining revealed that the
percentage of proliferating cells was reduced in YH lesions compared
with AH lesions (P<0.02, Figure 2G
). Rare
BrdU-positive nuclei were present in the intima of AC lesions.
Apoptotic cells as detected by TUNEL were rare. They were more
frequent in AH than in YH lesions (P<0.02, Figure 2H
). In AH lesions, rounded and elongated positive nuclei could
be detected, suggesting the presence of both macrophagic and myocytic
apoptotic cells (Figure 3H
). Very rare apoptotic
cells were observed in the tunica media of the AH (0.17±0.03%)
and YH (0.15±0.04%) groups. These values did not differ from those of
respective normolipemic controls. No apoptotic intimal cells
were detected in examined AC sections. To confirm the presence of
apoptosis, we used a modified ligation-mediated PCR. The use of
blunt-end linkers in this procedure allows us to amplify and quantify
the DNA ladder that is not distinguishable when standard genomic DNA
gel electrophoresis is used.37 Quantification of
ligation-mediated PCR products on agarose gel (Figure 7
) showed that the integrated optical
density value of AH sections was greater than that of YH sections
(P<0.01). No significant differences were observed when
integrated optical density values were compared in AC and YC aortic
tissue.
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| Discussion |
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It is evident that the diffuse aortic intimal thickening, as a simple favorable site for the development of fatty streaks, is not enough to explain the increased severity of lesions observed in aged rabbits after a long-term hypercholesterolemic diet.15 It is likely that other age-related intimal changes play an additional atherogenic role. We observed increased GAGs in AH lesions compared with YH lesions and AC intima and the scarcity of GAGs in YC subendothelial space, suggesting a preexisting plasma cholesterolindependent and age-related accumulation of GAGs.42 Because relative volume and cellularity did not differ, the increased amount of GAGs in AH lesions seems balanced by the hypertrophy of macrophagic FCs that constitute the majority of cells in YH lesions. GAGs are of great importance in influencing arterial properties, including permeability.43 Ultrastructural studies revealed that GAGs decorate blocks of amorphous and fibrin-like material inside AH insudate, suggesting a local role of GAGs in the pathogenesis of insudate. Circulatory plasma-derived molecules and lipids accumulate in regions of blood vessels with high concentrations of GAGs.43 The intense brilliant red staining of insudate by Movats pentachrome indicates the presence of fibrin and plasma-derived material accumulations. The association of plasmatic molecules and GAGs may promote the development of lipoprotein-GAG complexes44 and the clearance of plasma-derived lipid particles (in particular, triglyceride-rich VLDL) by intimal SMCs.45 In addition, inside insudate may accumulate other plasmatic atherogenic components, such as the terminal complex of complement,46 favoring the progression of lesions. The intimal accumulation of GAGs in the AC group does not appear as the result of a generalized aging process of the arterial wall, because it was more marked than that of the underlying tunica media. Intimal SMCs displayed a "synthetic" phenotype.47 Similar to experimental models of intimal thickening,48 49 50 they may derive migration and proliferation of specific subsets of medial SMCs into the intima with aging. The presence of GAGs associated with SMC exocytotic cytoplasmic vesicle of AH lesions suggests that SMCs further synthesize GAGs when subjected to an atherogenic stimulus.43 The relevant role of SMCs is strengthened by the finding of early fibromuscular caps in AH fatty streaks. The latter may contribute to accelerate the progression of lesions by limiting the survival of FCs entrapped below. Early and large accumulation of extracellular lipidic-necrotic material favors the development of an atheromatous core and the progression of some fatty streaks to atheromatous plaques.20 It is evident that age-related accumulations of GAGs could not explain by themselves the plasmatic insudate in AH lesions. Serofibrinous insudates have been reported in Watanabe or hypercholesterolemic New Zealand rabbits41 as well as in humans.2 Rosenfeld and Ross41 have suggested that insudates are the consequence of endothelial retractions after the hypertrophy of bulging subendothelial FCs after a marked hypercholesterolemic stimulus. The insudate that we observed is not the result of the same phenomenon because in the presence of a similar mild hypercholesterolemia, they were absent in YH lesions in spite of a more irregular lesion surface. AH insudate may derive from an exaggerated arterial transmural flux of plasmatic macromolecules in response to hypercholesterolemia. An age-related progressive thickening and fibrosis of the arterial wall51 and/or alterations of endothelial function may contribute to the development of insudate. SEM demonstrated an increase of monocytes adhering to endothelial surface areas far from the ostia similar to that found in aged rats,12 with an absence of variations of plasma lipemic parameters compared with conditions found in young rats. This suggests an intrinsic age-related dysfunction of endothelial cells.12 To confirm this hypothesis, we observed in the same sites a reduced eNOS expression, similar to that in aged rats.52 An impaired eNOS activity is considered a marker of endothelial dysfunction and one of the possible mechanisms leading to the reduction of endothelial levels of NO with aging.52 53 NO plays a relevant role in the maintenance of vascular homeostasis.52 53 A chronic inhibition of NO production accelerates neointimal formation and impairs endothelial function in hypercholesterolemic rabbits.54 An age-related reduction of NO production may predispose blood vessels to atherosclerosis55 and dramatically influence the transport properties of the rabbit aortic wall.56 Age-related endothelial dysfunction amplifies the hypercholesterolemia-induced decrease of eNOS bioavailability and may increase arterial permeability, thus favoring the accumulation of plasmatic substance and the development of lesions also in those areas that are nonsusceptible in young rabbits. Increased adhesion of circulatory monocytes and impaired eNOS levels may also be related phenomena, because NO gene therapy reduces adhesion molecular expression and, consequently, inflammatory cell infiltration.57
We also observed an increased intimal cell proliferative rate in AH compared with YH lesions. Besides confirming that cell proliferation is a phenomenon present during early phases of rabbit atherogenesis,58 the age-related higher cell proliferation rate contributes to the progression of lesions by increasing the intimal cell population in vivo19 20 as in other experimental conditions.22 23 24 In aged rats subjected to a hypercholesterolemic stimulus, aortic intimal cells, compared with underlying medial SMCs, associated phenotypic changes with a double proliferation.59 Preliminary studies by double immunohistochemistry demonstrated that macrophagic cells mainly proliferate in early as well as in advanced lesions of cholesterol-fed young rabbits, whereas in aged rabbits, myocytic and macrophagic cells proliferate (L.G.S. et al, unpublished data, 1999). The cell proliferation rate in AH lesions was similar to that previously reported in advanced plaques of aged rabbits after long-term cholesterol feeding.26 This differs from young cholesterol-fed rabbits, in which the proliferative index has been reported to decline with time.41 All these data suggest a double effect of aging: increasing the fatty streak proliferative rate and maintaining it at high levels during the progression to advanced plaques. Apoptosis also contributes to the modulation of the size of the intimal cell population.60 Apoptosis was more frequent in AH than in YH lesions, suggesting that this is an age-related phenomenon. We could clarify whether the modulation of apoptosis is intrinsic to the arterial wall of aged animals or is mediated by other conditions. However, the absence of apoptotic cells but not of proliferating cells in AC intimal tissue confirms that mechanisms regulating programmed cell death differ, at least in part, from those inducing growth arrest.61 62 The presence of apoptotic cells in AH fatty streaks excludes the possibility that they represent an adaptive intimal thickening.63 Humoral and cellular factors (in particular, locally delivered inflammatory cytokines) contribute to the control of apoptosis.61 64 We documented the presence of T-lymphocyte infiltration in AH and YH lesions, confirming this to be an early event in rabbit atherogenesis.58 T-lymphocyte infiltration was greater in AH than in YH lesions. This difference was sharper considering the lymphocyte percentage per total mononuclear cells, with the number of nonmyocytic cells being lower in AH fatty streaks. In YH lesions, lymphocyte percentage was similar to that reported for rabbits of comparable age and on similar diets.65 Because nonmyocytic cells were less prevalent in AH than in YH lesions, we conclude that the age-related increase of lymphocytes is selective and not dependent on a possible, spontaneous, generalized increase of adhering mononuclear cells to the endothelial surface with aging.13 66 Mechanisms responsible for this increased recruitment of T lymphocytes are still to be clarified. Because T lymphocytes have many relevant biological properties, including the secretion of inflammatory cytokines,67 it is likely that greater lymphocyte infiltrates deliver locally increased amounts of cytokines, thus favoring the progression of lesions.1 67 One may hypothesize that an age-related increase of lymphocytes contributes to the modulation of the intimal cell population of fatty streaks by triggering apoptosis. Successively, apoptotic cells may facilitate the progression to advanced plaques by further stimulating the adhesion of circulating monocytes and the activation of local factors, such as thrombin.68
In conclusion, the relation between aging and atherosclerosis appears to be a complex phenomenon. Parietal aging contributes to the development of fatty streaks in cholesterol-fed rabbits as a plasma-independent, prelesional, multifactorial risk factor. In addition, age-related influences on composition, proliferation, and apoptosis represent some of the mechanisms promoting the progression of fatty streaks to advanced plaques.
| Acknowledgments |
|---|
Received September 23, 1999; accepted October 7, 1999.
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