Thoracic aortic aneurysm (TAA) associated with Marfan syndrome (MFS) and
bicuspid aortic valve (BAV) is characterized histologically by vascular smooth muscle cell
(VSMC) loss, areas of cystic medial necrosis (CMN), patchy elastic lamellar (EL) destruction
and increased expression of matrix metalloproteinases (MMP)-2 and-9. MFS is an autosomal
dominant disorder of connective tissue caused by mutation of the FBN1 gene encoding
fibrillin-1 protein with a prevalence of about 0.2% regardless of race. BAV is the most
common congenital cardiac malformation, affecting 1-2 % of the population with a male
predominance. The cause of disease is unknown but it may affect multiple family members
across generations, suggesting an underlying genetic mechanism.
VSMCs are a major cellular component of the aorta responsible for synthesis and secretion of
collagen, elastic fibres and various types of proteoglycans. Premature apoptosis of VSMCs in
the tunica media of aortic aneurysm and an increased rate of apoptosis in cultured VSMCs
derived from MFS and BAV patients has been previously reported. Intracellular accumulation
of proteins (fibrillin, fibronectin, tenascin) in VSMCs obtained from MFS and BAV patients
has suggested abnormalities in cytoplasmic organelles responsible for synthesis and secretion
of protein (rough endoplasmic reticulum (RER) and Golgi complex) are a factor in the
pathophysiology. Increased synthesis of the gelatinases MMP-2 and MMP-9 in VSMCs
contributes to degeneration of the tunica media, loss of its structural integrity, and TAA
development. MMP-10 (stromelysin-2), another member of the MMP family, has recently been
found to be up-regulated in histone deacetylase 7 (HDAC-7) mutant mice and was associated
with destructive remodelling of the vascular ECM, weakened cell-matrix bonding and
consequent dilatation and rupture of blood vessels. As suggested, MMP-10 may participate in
initiating activity in the MMP cascade and in concert with other MMPs have a role in normal
tissue remodelling and pathological tissue destruction. Distribution, expression level of MMP-
10 and its possible role linking to alterations of VSMCs in MFS and BAV aneurysm however
have not been previously investigated.
Although destruction of tunica media structure is a common finding in aneurysm tissue the
primary cause that initiates the destructive changes has not been identified. In this study it was
hypothesised that intrinsic abnormalities of VSMCs lead to overexpression of MMP-10 and
alterations in cell-extracellular matrix (ECM) contact as well as abnormalities in physical
structure and function of elastic and collagen fibres. Such changes lead to VSMCs prone to
apoptosis and create favourable conditions for aneurysm formation and progression.
The aims of this thesis were: 1. to examine the morphology of VSMCs and their relationship
with ELs and the surrounding extracellular matrix (ECM) under light microscopy (LM) and
transmission electron microscopy (TEM); 2. to define the ultra-structure of VSMCs in culture,
focusing on cytoplasmic organelles associated with protein synthetic pathways; 3. to identify
the distribution and level of MMP-10 expression both in aortic wall and in cultured VSMCs as
well as to measure MMP-10 activity and MMP-10 quantity in conditioned media of cultured
VSMCs; 4. to evaluate the relationship between MMP-10 expression and medial cell depletion
in MFS and BAV aneurysm; 5. to detect VSMC loss by activated caspase-3.
Materials and methods: Thoracic aortic tissue and cultured VSMCs derived from normal
subjects and subjects with MFS or BAV aneurysm were used in this study. LM and TEM were
applied to examine the ultrastructure of aortic wall tissue and VSMCs in culture.
Immunohistochemical staining (IHC) for MMP-10 was performed to identify localization and
MMP-10 expression both in aneurysm wall and in cultured cells. Zymography and Western
blot was carried out to measure MMP-10 expression and activity. Positive staining for caspase-
3 by IHC was used as a marker to confirm identification of apoptotic cells.
Results: LM confirmed the presence of severe structural abnormalities of VSMCs that were
independent of areas of cystic medial necrosis (CMN). There were many morphologically
altered VSMCs (atypical cells). These cells were partly disintegrated and contained numerous
vacuoles and structural masses which occupied most of the cytoplasm in the area of intact EL.
Percentage of atypical cells was significantly higher in patient groups (MFS=34.0±0.1%*;
BAV=29.1±0.1%*, P<0.05) compared to normal subjects (10.1±0.1%). The proportion of
apoptotic cells in aneurysmal sections was significantly higher compared to controls
(normal=1.4±0.1%; MFS=8.3±0.1%*; BAV=10.0±0.1%*, P<0.05). TEM showed EL less
wave-like, thinner and with smoother surfaces. ELs were accompanied by a limited number of extensions to lock with neighbouring VSMCs. VSMCs showed rounder morphology, severe
degeneration, and were separated from nearby ELs and other surrounding cells as a result of
shorter or absent cytoplasmic projections. Severe morphological VSMC changes were found
commonly between intact ELs, consolidating the hypothesis that cell loss may occur before EL
fragmentation. Accumulation of ECM, cell debris, matrix vesicles, calcification of VSMCs and
ECM components were common findings in BAV.
For cultured VSMCs, under TEM, the mitochondria, RER and Golgi apparatus appeared
normal and showed no apparent differences between normal and MFS or BAV. The proportion
of apoptotic cells was significantly higher in MFS and BAV VSMCs compared to control
(control=2±3%, MFS=11±11%*, BAV=5±4%*; P<0.05), confirming the important role of
apoptosis in cell loss. Cultured VSMCs showed atypical cells (normal=14.8±0.1%;
MFS=38.8±0.1%*; BAV=29.0±0.1%*; P<0.05) with bulging intracellular masses, tearing of
the cytoplasmic membrane, cellular indentations and disintegration. These features were
similar to those recognized in aortic sections. Intracellular accumulations of proteins were
wrapped into vesicles or diffused throughout cytoplasmic region, resulting in complete
disorganization of the whole cell architecture. Numerous atypical cells were frequently
superimposed forming masses in culture which included debris of dead cells, severely
degenerate cells and large amounts of ECM. These findings suggest degeneration of cells as
another cause of diminished cell population. This is the first time such atypical cells have been
described in aortic tissue and cultured VSMCs of aneurysm disease, suggesting the reduction
of cell population is attributed to apoptosis and degeneration rather than apoptosis alone.
MMP-10 expression was assessed using colour imaging software. In tissue sections there was
enhanced expression of MMP-10 in VSMCs of aneurysm aorta compared to control subjects
(control=2.5±1.0; MFS=4.5±1.6*; BAV=4.7±1.5*, arbitrary units/VSMC/microscope field;
P<0.05). Similarly, MMP-10 staining in culture disclosed overexpression of MMP-10 in MFS
and BAV cultured VSMC (control=1.7±0.6; MFS=4.4±1.0*; BAV=6.1±1.2*, arbitrary
units/VSMC/microscope field; P<0.05), suggesting altered MMP-10 regulation in MFS and
The intensity of MMP-10 staining in individual VSMCs was classified as strong, mild, absent.
Strong expression of MMP-10 was observed in about 80% of apoptotic VSMCs
(control=77±30%, MFS=83±22%, BAV=78±15%) while F 20% of normal cells showed strong
signals of MMP-10 immunoactivity in cytoplasm (control=12±12%, MFS=20±14%,
BAV=14±10%). Increased MMP-10 expression was found to coincide with the increased
percentage of apoptosis in aortic tissue as well as in cultured cells. However, no active form of
MMP-10 was detected by gelatin and casein zymography in agreement with findings from
Western blot. Only proform expression of MMP-10 in VSMC conditioned media
(control=131±37; MFS=74±33*; BAV=64±36*; P<0.05) was identified using brightness scale
(higher number indicating lower expression levels). This indicates that MMP-10 was increased
in patient groups and was secreted in the latent form into the extracellular environment.
There was a high proportion of cell with caspase-3 staining in MFS and BAV aortic tissue
compared to control (control=10±3%, MFS=34±10%*, BAV=22±8%*) and a similar finding
in cultured cells (control=10±2%, MFS=30±8%*, BAV=28±3%*). Apoptotic cells which
showed condensation of nucleus were labeled either positively with caspase-3 or showed no
caspase-3 staining, providing information that the VSMC apoptotic pathway in TAA was either
caspase-3 dependent or caspase-3 independent. The proportion of cells with caspase-3 positive
staining was greater than the proportion of morphologically identified apoptotic cells in
control, MFS and BAV. In each of the 3 groups, most degenerate cells were strongly stained
with caspase-3 and a number of healthy looking cells showed caspase-3 signal in the
cytoplasm. This suggests that Caspas-3 may identify cells in earlier stages of apoptosis and/or
that caspase-3 also identifies atypical cells or cells undergoing degeneration. Furthermore, both
caspase-3 and MMP-10 were found strongly labeled in apoptotic cells and degenerate cells,
suggesting they may act in concert to initiate cell death.
Conclusion: The data indicate that morphological changes in VSMCs occurred before any
recognizable EL damage, supporting the hypothesis that the primary defect in the pathogenesis
of TAA arises within VSMCs. Characterisation of fragile connections between ELs and
VSMCs due to loss of elastic extensions and VSMC projections were an important finding.
These abnormalities are likely to play a fundamental role in the process of aneurysm formation.
Contrary to previous reports, the morphology of RER, Golgi apparatus and mitochondria
appeared normal in MFS and BAV patients. The abnormal presence of atypical cells both in
aortic tissue and in cultured cells indicates that this morphology is unique and inherent,
suggesting a novel pathogenic mechanism underlying VSMC degeneration and medial cell depletion. Although apoptosis continues to be an important cause of cell loss severe
degeneration of VSMCs was identified for the first time as another source of reduced medial
cell density. Over-expression of MMP-10 and increased activation of caspase-3 may contribute
to ECM remodelling, activation of the MMP cascade, disruption of cell attachments to ECM,
and weakened cell-matrix bonding with loss of medial cells during aneurysm progression.