Vascular Smooth Muscle Cell Apoptosis and Matrix Membrane Metalloproteinases in Thoracic Aortic Aneurysm

Jalali, Leyla (2007). Vascular Smooth Muscle Cell Apoptosis and Matrix Membrane Metalloproteinases in Thoracic Aortic Aneurysm Master's Thesis, School of Medicine, University of Queensland.

       
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Author Jalali, Leyla
Thesis Title Vascular Smooth Muscle Cell Apoptosis and Matrix Membrane Metalloproteinases in Thoracic Aortic Aneurysm
School, Centre or Institute School of Medicine
Institution University of Queensland
Publication date 2007
Thesis type Master's Thesis
Supervisor Professor Malcolm West
Abstract/Summary Background: Thoracic aortic aneurysm (TAA) associated with Marfan syndrome (MFS) and bicuspid aortic valve (BAV) shows elastic fibre fragmentation, up-regulation of matrix metalloproteinases (MMP) and vascular smooth muscle cell (VSMC) apoptosis with an absence of inflammatory cells. Recently, apoptosis has come to be considered as an important factor in VSMC loss during aneurysm formation. However, it is uncertain whether apoptosis happens secondary to aneurysmal formation or whether fundamental abnormalities in VSMCs lead to apoptosis as a primary cause in aneurysm formation. Therefore the exact stimuli for VSMCs apoptosis are not clear. Given the role of MMPs in matrix remodelling during morphogenesis and tissue repair, their involvement in aortic aneurysm has been frequently suggested. Abnormality of the extracellular matrix influences elasticity of the aortic wall and may lead to disorganisation and weakening of the aortic wall, producing aortic dilation followed by aneurysm formation. Amongst all MMPs, matrix membrane metalloproteinase type 1 (MT1-MMP), an active cell membrane collagenase involved in cell migration and tissue remodelling, has commonly been documented to be associated with apoptosis. Another member of this family, MMP-2 (gelatinase A) activation is critically dependent on its processing by MT1-MMP. In addition MMP-2 has commonly been associated with apoptosis and aneurysm formation. This thesis has examined the association of VSMC apoptosis, MT1-MMP expression and MMP-2 activity in TAA. Methods and results: Studies were carried out on tissue specimens and cultured cells derived from aortic tissue obtained from subjects undergoing surgery for TAA: MFS (3M, 2F; mean age: 59 years) and BAV (5M; mean age: 64 years). Normal aortic tissue was obtained from organ donors (4F, 1 unknown, mean age: 43 years). The concentration of apoptotic cells in tissue and in cell culture was determined using the terminal deoxynucleotidyl transferase (TdT)-mediated nick end labelling (TUNEL) method to identify apoptotic cells. The concentration of MT1-MMP expressing cells in tissue and in cell culture was determined using immunochemistry. Gelatine zymography was performed to detect MMP-2 activity. Apoptotic cells (% of VSMC showing positive TUNEL marker/ high power field, mag 200) and MT1-MMP expression (% of cells showing positive MT1- MMP marker/ high power field, mag 200) in aneurysm tissue and cultured VSMC (mag 400) were compared with controls. Withdrawal of serum from cell culture media was used to induce apoptosis and a caspase inhibitor (ApoBlock) was used to block apoptosis. Cultured VSMCs were plated at a density of 10,000 /ml in standard culture medium with 10% foetal calf serum (FCS), serum free media (absence of FCS) or serum free media + caspase inhibitor (ApoBlock) for 24 hours. The degree of apoptosis and number of MT1-MMP expressing cells were significantly increased in aneurysm specimens (P< 0.05) in both tissue and in cultured cells. Apoptosis level was 10x higher in MFS (21% ± 10%) and 6x higher in BAV (13% ± 7%) tissue specimens compared to normal controls (2% ± 1%, P< 0.05). Number of MT1-MMP expressing cells was increased in both MFS (31% ± 8%) and BAV (36 % ± 12%) in comparison with controls (1% ± 1 %, P< 0.05). In aneurysm derived cells cultured in standard media apoptosis levels and number of MT1-MMP expressing cells were also increased in MFS and in BAV compared to normal cells (P< 0.05). For cells cultured in serum free media (absence of FCS) apoptosis levels and number of MT1-MMP expressing cells were even more increased compared to control cells. The number of MT1-MMP expressing positive cells was significantly reduced in MFS (65% ± 7%) and BAV (47% ± 18%) VSMCs cultured in serum free media in the presence of caspase inhibitor compared to MFS (88% ± 8%, P< 0.05) and BAV (77% ± 17%, P< 0.05) cells cultured in serum free media only. The percentage of MT1-MMP positive cells identified in combined serum free media and caspase inhibitor however was greater than the percentage of MT1-MMP positive cells in normal culture media, indicating MT1-MMP was likely to be only partly inhibited. Both pro and active forms of MMP-2 detected in culture media extracted from BAV and MFS cells cultured in the presence and absence of caspase inhibitor were significantly higher compared to controls. However, there was no significantly difference in pro and active MMP-2 activity in cultured VSMCs in the presence of caspase inhibitor compared to cultured VSMCs in the absence of caspase inhibitor. Conclusion: There was significantly increased concentration of cells undergoing apoptosis and expressing MT1-MMP marker in aortic aneurysm tissue and in cultured cells compared to normal tissue or normal cells respectively. Under conditions of stress induced by serum free media VSMCs derived from aneurysm were significantly more vulnerable to apoptosis and showed a greater concentration of cells expressing MT1-MMP marker compared to cells grown in standard media. The concentration of MT1-MMP positive cells was significantly reduced in the presence of caspase inhibitor (ApoBlock) but did not reach the percentage level of MT1-MMP positive cells in standard culture conditions. MMP-2 activity was significantly higher in cultured media derived from aneurysm cells both in the presence and absence of caspase inhibitor. However, MMP-2 was not significantly reduced after blocking apoptosis pathway, indicating lack of a direct association between apoptosis pathway and activated MMP-2. MT1-MMP positive cell expression was significantly reduced by inhibiting apoptosis pathway, suggesting MT1-MMP is a contributing factor to VSMC apoptosis in aneurysms associated with MFS and BAV, although other contributing factors can not been excluded.

 
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