(Circulation. 1999;99:96-104.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Death of Smooth Muscle Cells and Expression of Mediators of Apoptosis by T Lymphocytes in Human Abdominal Aortic Aneurysms
From the Cardiovascular Division, Department of Medicine, Vascular Medicine and Atherosclerosis Unit (E.L.H., G.K.S., P.L.), and Division of Vascular Surgery, Department of Surgery (A.D.W., J.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Mass, and the Cardiovascular and Pulmonary Research Institute, Allegheny University of the Health Sciences, Pittsburgh, Pa (Y.-J.G.).
Correspondence to Peter Libby, MD, Vascular Medicine and Atherosclerosis Unit, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave, LMRC 307, Boston, MA 02115. E-mail plibby{at}rics.bwh.harvard.edu
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Abstract |
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Background—Thinning of the tunica media and rarefaction of smooth muscle cells (SMCs) characterize aneurysmal aortas. Apoptosis determines the cellularity and morphogenesis of tissue. Macrophages and T lymphocytes infiltrate the wall of abdominal aortic aneurysms (AAAs) and produce death-promoting proteins (perforin, Fas, and FasL). This study investigated whether apoptosis occurs in association with the expression of these proteins.
Methods and Results—We examined signs of apoptosis and
expression of death-promoting mediators in segments of AAAs from
patients undergoing elective repair (n=20). Anti–
-actin
immunostaining showed a reduced number of SMCs in AAAs.
In situ terminal transferase-mediated dUTP nick end-labeling (TUNEL)
showed higher levels of DNA fragmentation in AAAs than in controls
(n=5). The AAA walls contained more cells bearing markers of
apoptosis than normal aorta (P<0.05, Student's
t test). Double immunostaining
identified SMCs and macrophages as the principal cell types
displaying fragmented DNA. Immunohistochemistry revealed that AAAs but
not normal aorta contained CD4+ and CD8+ T
cells that expressed well-characterized cytotoxic mediators: perforin,
which produces membrane damage, and Fas, which acts by ligand-receptor
interaction. Double immunostaining also identified SMCs
that expressed Fas. Immunoblotting confirmed the
presence and, in the case of Fas, activation of these proteins in
aneurysmal tissue.
Conclusions—Many medial SMCs in AAAs bear markers of apoptosis and signals capable of initiating cell death. Apoptotic death may contribute to the reduction of cellularity and to the impaired repair and maintenance of the arterial extracellular matrix in AAAs. Macrophages and T lymphocytes infiltrate the wall of AAAs, where they can produce cytotoxic mediators such as cytokines, perforin, and Fas/FasL. These death-promoting products of activated immune cells may contribute to elimination of SMCs, a source of elastin and collagen, during the pathogenesis of AAAs.
Key Words: aneurysm • muscle, smooth • cells • lymphocytes • apoptosis
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Introduction |
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Aneurysms have been recognized causes of morbidity and mortality for centuries. Descriptions of "pulsatile masses" and the consequences of their injury appeared in the Ebers Papyrus (ca 2000 BC) and the works of Galen (131 to 200 AD).1 Epidemiological investigations have revealed a 3% prevalence of aneurysms in persons
60 years old.1 2 3 The past half century has
provided a wealth of data concerning the natural history of
aneurysm formation. Investigations of the pathogenesis
of this disease, aimed at identification of a specific genetic defect,
have so far yielded no clear pathogenic mechanism of formation.
Although aneurysms can develop in any artery, the most striking
morphological alterations occur in the abdominal aorta and other large
arteries. Aortic aneurysms weaken and distort
arterial architecture and, in certain patients,
progressively enlarge and rupture. In late-stage aneurysms,
aortic structure changes, displaying breakdown of elastic laminae and
disappearance of well-organized smooth muscle layers. Smooth muscle cells (SMCs), the most abundant cell type in the aortic media, can synthesize proteins such as collagen, elastin, laminin, gelatin, and proteoglycan, which compose the extracellular matrix of the arterial tunica media and confer strength and elasticity to the aortic wall. A loss or distortion of these structural proteins characterizes aneurysms. Researchers have sought enzymes capable of causing such changes. The primary candidates have been matrix metalloproteinases (MMPs) and certain serine proteases. Although many investigators have noted an increase in the levels of MMPs in aneurysmal and nonaneurysmal atherosclerotic tissue, recent evidence of elevation of both MMPs and their inhibitors (TIMPs) in aneurysmal tissue suggests that the overall balance of enzyme activity may be preserved.4 Thus, although proteolytic degradation of extracellular matrix may contribute to the structural changes found in the aneurysmal arteries, other mechanisms may pertain. For example, a decrease in number of SMCs could impair synthesis of the matrix proteins needed for repair. However, little information has been available regarding mechanisms that might deplete SMCs in these lesions.
We tested the hypothesis that local expression of death-promoting mediators by infiltrating immune cells may promote the formation of abdominal aortic aneurysms (AAAs), which are notable not only for the decrease in SMCs but also for an inflammatory infiltrate. The abundance of lymphocytes and macrophages in these lesions suggests an ongoing, dynamic process of vascular remodeling. T lymphocytes initiate cell death by 2 pathways that involve distinct proteins, Fas and perforin. Fas is a member of the tumor necrosis factor (TNF) receptor family of membrane proteins. On binding Fas ligand (FasL), Fas initiates a cascade of signaling events that can kill the target cell. Activation of a series of proteases (caspases) figures prominently in this pathway. We have previously documented coexpression of the prototypic caspase, interleukin (IL)-1ß–converting enzyme, with SMCs bearing markers of apoptosis in human atheromata.5 The second death-promoting pathway of T lymphocytes uses perforin. When released from storage granules in cytotoxic T cells, perforin creates holes in the membranes of target cells that alter permeability and lead to cell death.6 7
Apoptosis contributes to normal morphogenesis.8 9 Recent studies have furnished evidence for apoptosis of SMCs in carotid and coronary atheromata as well as AAAs.5 10 In addition, cytokines produced by activated T cells and macrophages can cause the death of cultured human SMCs.11 The present study provides a novel potential mechanistic link between SMC death triggered by activation of the immune cells and weakening of the aortic wall. These changes may contribute to the pathogenesis of aneurysms and other aspects of arterial remodeling.
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Methods |
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Aneurysmal and Normal Aortic Tissue
Human AAA segments were obtained from patients undergoing elective repair (n=20). The average age was 70.6 years (range, 53 to 81 years). The average size of the aneurysmal lesions estimated by CT scan and/or angiography was 5.8 cm (range, 4.5 to 8.0 cm). Eighty percent of patients were men and 20% women. Nonaneurysmal aortic specimens were obtained at autopsy from 5 subjects who had no evidence or medical history of aneurysmal or occlusive disease (average age, 61.6 years; range, 46 to 84 years; 60% women, 40% men). Normal aortic tissue obtained from organ donors or from the collection of aortic tissue in the Pathological Determinants of Atherosclerosis in Youth archive was used as control. Fresh aneurysmal tissue was divided into 2 parts: 1 for frozen or paraffin sections for histochemistry or immunohistochemistry and detection of apoptotic cells and 1 for protein isolation. The study of normally discarded human tissue was approved by the Human Investigation Review Committee at Brigham and Women's Hospital.
In Situ End-Labeling of DNA Fragments (TUNEL)
Limited fragmentation of genomic DNA characterizes
apoptosis. To evaluate numbers of cells with such damage to
genomic DNA, we performed in situ labeling of DNA fragments by use of
TUNEL with an ApopTag in situ apoptosis detection kit
(Oncor, Inc). Briefly, sections were deparaffinized, rehydrated, and
incubated with 0.3% H2O2
in PBS to quench endogenous peroxidase activity. The slides
were then incubated with 20 µg/mL of proteinase K (EM Science) and
10 mmol/L EDTA in PBS. DNA fragments were labeled with
digoxigenin-dUTP. The terminal transferase and the labeled DNA
fragments were detected with peroxidase-conjugated antibody against
digoxigenin. Diaminobenzidine (DAB) was used as the substrate for
peroxidase, yielding a brown color in nuclei. The sections of AAAs
(n=20) and the controls (n=5) were viewed via light microscopy. For
each section, the cells of 10 contiguous fields were counted
independently by 2 investigators, and their observations were averaged.
Cells with visible nuclear condensations were counted as
positive.12 Apoptotic index was determined by the
formula 100x(number of TUNEL-positive cell nuclei/total number of cell
nuclei). Using parallel slides, we stained for -actin
(HHF-35). The same investigators counted the number of SMCs
present in the wall of normal and aneurysmal aortas. A
second index was determined with the same numerator and a denominator
of -actin–positive SMCs. The number of nuclei per cross-sectional
area was calculated from the formula (perimeterxthickness)xnumber of
nuclei/high-power field divided by the area of the high-power field.
The perimeter was the average aortic diameter (normal, 2.3 cm; AAA, 5.8
cm), average aortic wall thickness (normal, 0.25 cm; AAA, 0.75
cm).
Immunohistochemistry
Cellular markers and products of immune cells in AAAs and
control tissues were analyzed by immunohistochemistry with
monoclonal antibodies (Table 1
).
Serial cryostat sections (6 µm) were prepared and air-dried on
poly-L-lysine–coated slides. After fixation in acetone at
-20°C for 5 minutes and pretreatment with 0.3%
H2O2 and proteinase K (EM
Science), sections were incubated for 20 minutes in a blocking solution
of 5% normal horse serum and then stained for 1 hour with a panel of
primary antibodies (Table 1). Sections were washed and incubated
with biotin-conjugated horse antibody (Vector Laboratories, Inc), which
recognizes mouse IgG (1:200), for 30 minutes at room temperature. An
avidin-alkaline phosphatase-fast red reagent (Vectastain ABC kit,
Vector) visualized bound antibodies. Normal mouse IgG (Sigma Chemical
Co) was used as the control for the immunostains. For
double staining, sections were first stained by TUNEL for detection of
DNA fragments and then by immunohistochemistry for determination of
cell-specific antigens. Four hundred cells were counted for each
section by 2 independent observers.
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Immunoblot Analysis
A portion of the tissue samples was snap-frozen, crushed in
liquid nitrogen, and mixed with 0.5 mL of SDS protein extraction buffer
(20 mmol/L NaCl, 100 mmol/L Tris-HCl, pH 7.6, 10% SDS).
After centrifugation at 4°C for 20 minutes at
13 000g, the supernatant was collected and protein
concentration determined with BSA as a standard. Proteins (30
µg/lane) were loaded into a 12.5% SDS-PAGE minigel and separated at
100 V for 1.25 hours. For determination of activated Fas
aggregate, the samples were separated on a 7.5% SDS-PAGE gel. After
electrophoresis, proteins were transferred to a membrane by use of a
Trans-Blot SD semidry electrophoretic transfer cell (Bio-Rad). The
membranes were blocked with a 5% fat-free milk solution and incubated
in PBS with primary antibodies (Table 1
) against different
T-cell antigens overnight. After staining, the membranes were washed in
PBS with 0.2% Tween 20 and incubated with horseradish
peroxidase–linked secondary antibody (goat anti-mouse, 1:10 000) for
1 hour. The membranes were washed again and developed with the
Renaissance Western Blot Chemiluminescence Reagent (Dupont) or enhanced
chemiluminescence system (Amersham).
Statistics
Significant differences between means were determined by
Student's t test. A value of P<0.05 was
considered significant.
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Results |
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Paucity of SMCs in AAAs
In normal aortic media, almost all SMCs showed immunoreactive
-actin and formed homogeneous layers separated by
elastic laminae in an orderly pattern (Figure 1a and 1b). In contrast, the
aneurysmal aorta exhibited fewer actin-positive cells (Figure 1c and 1d). Certain areas of AAAs appeared hypocellular, with
little actin immunostaining. In the AAA regions with a
relatively higher cell density, SMCs were often fragmented and adjacent
to areas of acellular matrix (Figure 1c and 1d). These cells
displayed weak immunoreactivity toward HHF-35, indicating a lower
content of -actin. AAAs lacked the orderly structure of elastic
laminae observed in normal aortic media and showed a haphazard array of
remaining SMCs interspersed with mononuclear leukocytes (Figure 1c and 1d). Cell counting revealed a 44% (25% to 66%)
reduction in SMC density in AAAs (n=29) compared with the normal
controls (n=5) (Table 2). To evaluate
whether there was an actual decrease in the absolute number of SMCs in
the aneurysmal wall as opposed to a similar number of cells
dispersed over the enlarged aneurysmal wall, we calculated the
number of nuclei per cross-sectional area. This calculation showed a
36% decrease in the nuclei per unit area in AAAs compared with that of
normal aortas.
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Infiltration of Inflammatory Leukocytes
We examined the aneurysmal tissue for
monocyte/macrophages or lymphocytes by immunohistochemistry
(Table 1). The media of normal control aortas contained few
CD68+ macrophages or
CD3+ T cells. However, aneurysmal tissue
showed focal accumulation of monocyte/macrophages, T
lymphocytes, and to a lesser degree, but still above baseline control
levels, B cells and natural cells (histology not shown). To clarify
which subsets of T cells existed in the arterial tissues,
we stained the sections with antibodies that recognize the CD4 and CD8
antigens characteristic of T helper and T cytotoxic cells,
respectively. We found both CD4+ and
CD8+ T-cell subsets in the aneurysms
(Figure 2). We found more
CD4+ cells than CD8+ cells.
Negative controls using nonimmune IgG showed no positive staining
(Figure 2d). These immunohistochemical results indicate
coexistence of aortic degeneration and infiltration by inflammatory
cells in AAAs.
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AAAs Exhibit Increased Markers of Apoptosis
The lower number of SMCs in aneurysmal aortic walls
suggested that cell death might mediate elimination of SMCs. To test
this hypothesis, we analyzed DNA fragmentation using the TUNEL
technique. Nonaneurysmal aortic tissue contained few
TUNEL-positive cells (<1%) in the media (Figure 3a and 3b), whereas AAAs contained
numerous TUNEL-positive cells. Cells bearing this marker of
apoptosis localized within the lesions with inflammatory
infiltrate, particularly in the region of intima-media junction (Figure 3c and 3d). Double staining with a combination of TUNEL and
immunohistochemistry using antibodies against SMCs and
macrophages determined which cell types bore this
apoptotic marker (Table 1). Both -actin–positive
SMCs and CD68-positive macrophages were TUNEL-positive (Figure 3). Cell counting revealed an increase in apoptotic
index in AAAs (6.78±3.1 SD) compared with normal tissue
(0.57±0.3 SD) (Table 2). To ensure that the index was not
skewed by the absence of cells in the aneurysms, a second index
was calculated using only SMCs as the denominator (Figure 4), which demonstrated
consistently increased numbers of SMCs with TUNEL-positive
nuclei in AAAs (13.5±6.2 SD) versus control (no appreciable change
from noncorrected index, SD).
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Candidate Mediators of Cell Death in AAAs
Further studies explored the nature of sources of signals that
might trigger cell death in AAAs. The normal aorta contains little Fas
antigen, limited to regions of the intima, with occasional
Fas+ cells in the media (Figure 5). In contrast, AAA tissue showed
elevated levels of Fas throughout, prominently in both SMCs and
leukocytes within the media. Double immunostaining of
AAA specimens with -actin and Fas demonstrates that SMCs express
this receptor (Figure 5). Many TUNEL-positive cells also stain
for Fas. AAA lesions but not control aortas contained FasL-positive T
cells (histology not shown). Colocalization of both Fas and its ligand
in AAA tissue suggested operation of the Fas death-signaling pathway.
We sought evidence that the T cell–derived effector of cell death,
perforin, might also participate in cell death within AAAs. Normal
aortas contained little or no perforin, but in AAAs, immunoreactive
perforin colocalized with activated T cells (Figure 6).
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Immunoblotting evaluated levels of perforin, Fas,
or FasL proteins in extracts of AAA or control tissue (Figures 7a through 7c and
8). To evaluate the specificity of the
immunoblots, a negative control was incubated in blocking
media and secondary antibody alone (Figure 7d).
Consistent with the data obtained by immunohistochemistry, AAAs
but not normal aortas contained appreciable amounts of perforin, Fas,
and FasL (Figure 7a through 7c). Recent work by Kamitani et
al13 has shown that certain anti-Fas antibodies recognize
activated aggregates of human Fas. Indeed,
immunoblots using these antibodies (3D5 and G254-274)
disclosed higher-molecular-weight Fas aggregates, particularly a 97-kDa
fragment in extracts of AAAs but not normal aortas, indicating Fas
activation in the diseased tissue (Figure 8).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Exploration of the pathogenesis of aneurysmal disease has evolved considerably over the past 30 years. Early studies focused on prevalence and natural history, attempting to define risk profiles for developing aneurysms and to determine whether additional factors might precipitate rupture.1 3 14 15 The association of some aneurysms with connective tissue disorders led to genetic evaluations of families with multiple members affected and to the search for culprit genes.1 2 16 17 Experiments have sought to identify enzymes in the aorta that might break down collagen and elastin, which compose major structural elements of the vessel wall. Recently, Knox et al4 compared MMP expression in aneurysmal and occlusive aortic diseases with normal controls and found no difference in MMP or TIMP expression that would explain aneurysm formation versus occlusive disease. It remains obscure why patients with seemingly identical risk profiles develop opposite manifestations of atherosclerosis.
Lack of SMCs, the source of the arterial extracellular matrix, might favor aneurysm formation. Cell death occurs in nonaneurysmal atherosclerosis.5 18 19 Recent studies have shown evidence of apoptosis of SMCs in AAAs. Because loss of SMCs might contribute etiologically to aneurysmal disease, we sought the potential mechanisms of this process.
A paucity of SMCs in AAAs compared with the accumulation of SMCs in
occlusive disease is one of the most striking
histological distinctions between these 2 conditions.
Histological examination of AAAs consistently
shows distinct loss of cellular structures and nuclei, arguing against
a substantial population of -actin–negative SMCs.
The present results with perforin and Fas/FasL point to a role for
immune cells in induction of apoptosis of SMCs in
aneurysmal tissue.20 In situ observations cannot
provide direct evidence for this possibility. However, our recent in
vitro work has demonstrated that SMCs can undergo apoptosis
triggered by activation of Fas after priming with proinflammatory
cytokines such as interferon-
, TNF, and IL-1.11
Ligation of Fas caused aggregation, reflected by the appearance of
higher-molecular-weight bands in
immunoblots.13 Our observation that such
97-kDa Fas aggregate fragments exist in AAA but not normal aortic
tissue provides further biochemical evidence for the role of the
Fas/FasL system in cell death in AAAs. Macrophages and T cells
within evolving aneurysms most likely produce these
cytokines locally; these mediators may act in concert with
Fas/FasL to induce SMC apoptosis. The low levels of
inflammatory cells in normal aorta probably explain the lower
expression of Fas/FasL and perforin than in AAAs, because T cells are
considered the primary source for these molecules. In addition to SMCs,
the inflammatory cells themselves may be targets for apoptosis,
a potential mechanism of self-limitation of the immune response.
The present data support the view that the pathogenesis of aneurysm formation as well as their distinction from aorto-occlusive disease may depend on the inflammatory infiltrate, including T cells expressing FasL and perforin.
Not every T cell produces every cytokine, and activated
T cells can be subtyped according to their cytokine profile.
Th1 cells secrete IL-2, TNF-ß, and IFN-, whereas Th2 cells
typically produce IL-4, -5, -6, and -10. Because Th1 cytokines
can promote apoptosis in SMCs, Th1 cells may mediate loss of
SMCs in AAAs. Conversely, the cytokines elaborated by Th2 cells
may promote a less cytolytic response that could culminate in the
clinical picture of aorto-occlusive disease. Continuing studies should
further characterize the T cell subtypes and the cytokines
present in the 2 diseases.
It is important to acknowledge the limitations of a study that uses
surgically removed human aneurysmal vessels. AAAs are largely
asymptomatic until they have reached a size that requires
intervention. This usually precludes obtaining fresh specimens from
other than end-stage disease. Therefore, we cannot extrapolate from
observations on late-stage specimens to earlier phases of
aneurysm formation. In particular, the high apoptotic
index for SMCs (Figure 4) may not reflect an actual
instantaneous rate of apoptosis. As previously stated, care was
taken during the cell counting to ensure that condensed nuclei were
identified, avoiding the false-positives generated by staining of
calcium-phospholipid vesicles.12 However, as recently
reported, cells undergoing RNA splicing may stain positive with TUNEL,
rendering this technique selective but not specific, because
apoptotic nuclei contain an increased degree of fragmentation
compared with nonapoptotic cells.21 Existing
animal models use exposure of arteries to exogenous enzymes or
xenografting of arterial tissue, 2 procedures that may not
mimic the usual pathogenesis of human aortic
aneurysms16 (E. Allaire, MD, unpublished
data, 1997). These limitations make it difficult to draw conclusions
regarding signaling and initiation of events in relation to the human
disease. The present identification of 2 signals that may be
involved in altering SMC function in human aortic aneurysms may
aid in the development of an improved animal model for this important
arterial disease.
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Acknowledgments |
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Dr Henderson was supported by Harvard Longwood Basic Science Vascular Surgery Training Grant T32-HL-07734. Dr Libby is supported by NIH/NHLBI grants HL-43364, HL-48743, and HL-34636. We would like to thank Dr Todd Bourcier for his helpful advice. We also thank Eugenia Schvartz for expert technical assistance.
Received July 23, 1998; revision received September 9, 1998; accepted October 7, 1998.
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 H. Li, S. Telemaque, R. E. Miller, and J. D. Marsh High Glucose Inhibits Apoptosis Induced by Serum Deprivation in Vascular Smooth Muscle Cells via Upregulation of Bcl-2 and Bcl-xl Diabetes, February 1, 2005; 54(2): 540 - 545. [Abstract] [Full Text] [PDF]
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K. A. Lindstedt and P. T. Kovanen Proteolysis of Pericellular Matrix: A Process Linking Inflammation to Plaque Destabilization and Rupture Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2205 - 2206. [Full Text] [PDF]
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J. C. Choy, V. H.Y. Hung, A. L. Hunter, P. K. Cheung, B. Motyka, I. S. Goping, T. Sawchuk, R. C. Bleackley, T. J. Podor, B. M. McManus, et al. Granzyme B Induces Smooth Muscle Cell Apoptosis in the Absence of Perforin: Involvement of Extracellular Matrix Degradation Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2245 - 2250. [Abstract] [Full Text] [PDF]
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E. Sho, M. Sho, H. Nanjo, K. Kawamura, H. Masuda, and R. L. Dalman Hemodynamic Regulation of CD34+ Cell Localization and Differentiation in Experimental Aneurysms Arterioscler. Thromb. Vasc. Biol., October 1, 2004; 24(10): 1916 - 1921. [Abstract] [Full Text] [PDF]
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J. Yang, K. Sato, T. Aprahamian, N. J. Brown, J. Hutcheson, A. Bialik, H. Perlman, and K. Walsh Endothelial Overexpression of Fas Ligand Decreases Atherosclerosis in Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., August 1, 2004; 24(8): 1466 - 1473. [Abstract] [Full Text] [PDF]
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 F.-X. Schmid, K. Bielenberg, S. Holmer, K. Lehle, B. Djavidani, C. Prasser, C. Wiesenack, and D. Birnbaum Structural and biomolecular changes in aorta and pulmonary trunk of patients with aortic aneurysm and valve disease: implications for the Ross procedure Eur. J. Cardiothorac. Surg., May 1, 2004; 25(5): 748 - 753. [Abstract] [Full Text] [PDF]
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 C. NAPOLI Oxidation of LDL, Atherogenesis, and Apoptosis Ann. N.Y. Acad. Sci., December 1, 2003; 1010(1): 698 - 709. [Abstract] [Full Text] [PDF]
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T. S. Absi, T. M. Sundt III, W. S. Tung, M. Moon, J. K. Lee, R. R. Damiano Jr, and R. W. Thompson Altered patterns of gene expression distinguishing ascending aortic aneurysms from abdominal aortic aneurysms: complementary DNA expression profiling in the molecular characterization of aortic disease J. Thorac. Cardiovasc. Surg., August 1, 2003; 126(2): 344 - 357. [Abstract] [Full Text] [PDF]
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F.-X. Schmid, K. Bielenberg, A. Schneider, A. Haussler, A. Keyser, and D. Birnbaum Ascending aortic aneurysm associated with bicuspid and tricuspid aortic valve: involvement and clinical relevance of smooth muscle cell apoptosis and expression of cell death-initiating proteins Eur. J. Cardiothorac. Surg., April 1, 2003; 23(4): 537 - 543. [Abstract] [Full Text] [PDF]
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 J. S. Forrester Prevention of Plaque Rupture: A New Paradigm of Therapy Ann Intern Med, November 19, 2002; 137(10): 823 - 833. [Abstract] [Full Text] [PDF]
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Y.-J. Geng and P. Libby Progression of Atheroma: A Struggle Between Death and Procreation Arterioscler. Thromb. Vasc. Biol., September 1, 2002; 22(9): 1370 - 1380. [Abstract] [Full Text] [PDF]
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 U. Schonbeck, G. K. Sukhova, N. Gerdes, and P. Libby TH2 Predominant Immune Responses Prevail in Human Abdominal Aortic Aneu |