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Interact CardioVasc Thorac Surg 2008;7:37-40. doi:10.1510/icvts.2007.163311
© 2008 European Association of Cardio-Thoracic Surgery
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Work in progress report - Valves

Matrix metalloproteinase expression in the ascending aorta and aortic valve{star}

Emma Wiltona, Martin Blandb, Matthew Thompsona and Marjan Jahangiria,*

a Department of Cardiac Surgery and Cardiac and Vascular Sciences, St. George's Hospital, University of London, London, UK
b Department of Statistics, University of York, UK

Received 16 July 2007; received in revised form 30 October 2007; accepted 31 October 2007

{star} This work was supported by The Royal College of Surgeons of England by awarding E.W. a Research Fellowship for this study and by the St. George's Hospital Charitable Foundation.

*Corresponding author. Department of Cardiac Surgery, St. George's Hospital, Blackshaw Road, London, SW17 0QT, UK. Tel.: +44 208 725 3565; fax: +44 208 725 2049.

E-mail address: marjan.jahangiri{at}stgeorges.nhs.uk (M. Jahangiri).


   Abstract
Top
 Abstract
1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
 References
 
Extracellular matrix degradation and increased proteolytic enzyme (matrix metalloproteinase (MMP)) activity characterise abdominal aortic aneurysm formation. Post-stenotic dilatation of ascending aorta is associated with aortic stenosis and regurgitation, haemodynamically normal bicuspid aortic valve (BAV) and following AV replacement. We aimed to determine an association between ascending aortic pathology and abnormal AV, with particular reference to MMPs, and ascertain differences between BAV and tricuspid (TAV) AV. Subset of the study population (n=19) with a preoperative ascending aorta of >4 cm was analysed. Samples of ascending aorta and AV were obtained from 82 patients (TAV, n=54, BAV, n=28) undergoing surgery. Gene expression of MMP-1, -2, -9 and tissue inhibitor of metalloproteinase (TIMP)-1 and -2 was quantified by real-time RT-PCR. No significant difference was seen in gene expression level of MMPs, TIMPs and ratio of MMPs/TIMPs in ascending aorta and AV between patients with BAV and TAV. MMP-2/TIMP-1 in ascending aorta was greater in BAV, in the subset of patients with preoperative aortic dilatation (P<0.05). No difference exists in gene expression of MMPs in ascending aorta and AV between patients with BAV and TAV. However, patients with larger aortic diameters have increased MMP-2/TIMP-1. Modifying MMP expression may have a role in development of aneurysms.

Key Words: Aneurysm; Aorta; Aortic root; Metalloproteinases (MMPs); Valve disease


   1. Introduction
Top
 Abstract
 1. Introduction
2. Patients and methods
 3. Results
 4. Discussion
 References
 
The formation and expansion of an abdominal aortic aneurysm (AAA) is characterised by extracellular matrix degradation, increased proteolytic enzyme activity and an inflammatory cell infiltrate [1, 2]. We, and others, have shown that the balance of expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate the net degeneration of extracellular matrix and can lead to formation and rupture of AAA [3].

Post-stenotic dilatation of the ascending aorta is seen in patients with aortic stenosis (AS) and/or aortic regurgitation (AR), patients with a haemodynamically normal bicuspid aortic valve (BAV) and following aortic valve replacement (AVR) [4]. This dilatation is usually progressive, ≥0.3 cm/year. Aortic dilatation is thought to be a precursor to aortic dissection and rupture, both of which are potentially fatal.

It has been postulated that intrinsic pathology of the ascending aortic wall, involving an abnormality in the process of extracellular matrix remodelling, including inadequate synthesis, degradation and transport of extracellular matrix proteins, similar to that seen in AAA formation, may be the underlying mechanism of this dilatation of the ascending aorta.

In this study, we aimed to determine an association between ascending aortic pathology and abnormal AV, with particular reference to vascular wall matrix components that have been implicated in AAA formation using gene expression analysis. We also aimed to ascertain any difference in the matrix components of the ascending aorta and AV in patients with BAV and TAV.


   2. Patients and methods
Top
 Abstract
 1. Introduction
 2. Patients and methods
3. Results
 4. Discussion
 References
 
2.1. The study population

A prospective study of 82 consecutive unselected patients undergoing surgery for the AV and/or aorta between May 2005 and May 2006 was carried out. The mean age was 68.2±1.4 years with a male:female ratio of 1.8:1. The study population was subdivided into those with a native BAV defined as having two separate functional leaflets, and those with a native TAV, defined as having three separate functional leaflets. Patients presenting with infective endocarditis were excluded from the study. A subset of patients with preoperative ascending aorta of >4 cm was analysed.

Demographic data and result of preoperative transthoracic echocardiogram were collected.

Of the 82 patients, 73 underwent AVR, six had aortic root replacement and three had Yacoub's valve preserving procedure. The aortotomy was performed well above the sinus in the anterior aspect of the aorta, in the classic position of the AVR. Cardiopulmonary bypass was established in all cases by cannulation of the distal ascending aorta. None of the patients had dissection. Three (4%) patients were found to have a connective tissue disorder and subsequently diagnosed as having Marfan syndrome.

Ethical approval was obtained from the Ethics Committee. All samples were collected after written informed consent had been obtained.

2.2. Tissue acquisition and preparation

AV and a full-thickness biopsy of the ascending aortic wall, obtained at closure of the aortotomy, were collected at the time of surgery.

AV and ascending aortic specimen, once excised, were immediately submerged, separately, in RNA later RNA Stabilization Reagent (Qiagen, Hilden, Germany). The samples were then stored at –20 °C. These tissues were to undergo analysis by RT-PCR at a later date. This method of collection of human tissue samples, for analysis of gene expression, is recommended by the manufacturer of the RNA extraction kit, Qiagen.

2.3. Real-time reverse transcription-polymerase chain reaction

Analysis using quantitative real-time RT-PCR was undertaken. Relative gene expression level of MMPs (1, 2 and 9) and TIMPs (1 and 2) were measured.

2.4. Echocardiography

Transthoracic echocardiograms were performed preoperatively and postoperatively at one week, six weeks, six months and one-year follow-up.

2.5. Data and statistical analysis

Statistical analysis was performed using SPSSv12.0 for Windows (SPSS Inc., Chicago, IL). Results are expressed as mean±S.E.M. unless otherwise stated. Differences in the means between the two independent populations were compared using the unpaired Student's t-test or the Mann–Whitney U-test for continuous variables and the {chi}2-test or the Fisher exact test for categorical variables. P-values <0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
4. Discussion
 References
 
A total of 82 patients were entered into the study. All patients underwent preoperative transthoracic echocardiography.

Patients were separated into two groups on the basis of their native valve type [BAV (n=28) and TAV (n=54)] and the baseline demographic data examined (Table 1). There was no significant difference in gender, BMI, presence of hypercholesterolemia, connective tissue disorder, congenital heart disease, preoperative medication, diabetes and smoking between the two groups.


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  		Table 1 Demographic data for BAV and TAV

 
No significant difference was observed in the presence, or grade, of AS, AR and LV function preoperatively between patients with BAV compared to TAV (Table 1).

3.1. Real-time reverse transcription-polymerase chain reaction

The mRNA levels of selected genes, MMP-1, -2, -9, TIMP-1, and -2, in ascending aorta and the AV, were determined using qRT-PCR. 18S rRNA acted as an internal control and the level of expression of the genes of interest were compared to its level of expression (Table 2, Figs. 1 and 2).


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  		Table 2 Mean gene expression level of MMPs and TIMPs-BAV compared to TAV

 

Figure 1
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  		Fig. 1. Difference in gene expression between BAV and TAV.

 

Figure 2
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  		Fig. 2. (a) Comparison in degree of preoperative AS between BAV and TAV. (b) Comparison in degree of preoperative AR between BAV and TAV.

 
A subset involving 19 patients (mean age 67.8±3.9 years) with a preoperative dilated ascending aorta, diameter of ≥4.0 cm, was analysed. Gene expression levels of MMPs and TIMPs were compared. No significant difference was seen in the level of gene expression of MMP-1, -2 and -9 or TIMP-1 and -2 in the ascending aorta or the AV in patients with BAV (n=6) and TAV (n=13). However, a significantly greater ratio of MMP-2/TIMP-1 in the ascending aorta was observed in patients with BAV compared to TAV, in this subset (P=0.05).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
References
 
Hypertension was less common in patients with BAV. Galis et al. [5] showed that hypertension induces expression of MMPs in vascular tissues and this may be a reason for the similar levels of gene expression of MMPs between the mainly normotensive patients with BAV and the mainly hypertensive patients with TAV.

The MMPs are a family of proteases that play an important role in the homeostasis of connective tissue. They are synthesised by a variety of cell types, are secreted as pro-MMPs and can be activated by a number of proteinases including other MMPs [6]. Their production is regulated at the transcriptional level, and MMP activity is additionally regulated by proteolytic cleavage of inactive proenzymes to active forms and inhibition by TIMPs. The expression of TIMPs in tissue, like MMPs, is controlled during tissue remodelling and physiological conditions to maintain a balance in the metabolism of the extracellular matrix (ECM). The balance between MMPs and TIMPs regulates the degradation of the ECM in normal and pathological states. MMP expression and activity is regulated by haemodynamics, injury, inflammation and oxidative stress of vessels and brings about vascular remodelling [5]. The balance between MMP and TIMP expression regulates the net degeneration of extracellular matrix and can lead to aneurysm formation.

Studies have identified increased expression of MMP-1, -2 and -9, TIMP-1 and -2 and MMP/TIMP ratios as an important factor in the aetiology of AAA formation but the predominant MMP expressed in AAA is MMP-9 [7].

Ascending aortic aneurysms have previously been shown to exhibit increased protein level of MMPs, especially MMP-2, when compared to controls (non-aneurysmal aortic tissue) and the protein level also differed between aneurysms associated with BAV and TAV [8, 9]. We set out to analyse MMP and TIMP in a series of consecutive unselected patients, and not with established aneurysm, to ascertain whether there is a genetic element or haemodynamic factors present influencing the future development of ascending aortic aneurysm.

In our study, we found no difference in the gene expression level of the MMPs and TIMPs studied in the ascending aorta of patients with BAV and TAV. This may be because the ascending aortas were, in the majority, not dilated. Boyum and colleagues analysed the protein level of MMPs and TIMPs present in the ascending aorta rather than the gene expression level. The RT-PCR technique is highly sensitive but the RT-PCR reaction only gives an estimate of mRNA production for each of the enzymes and inhibitors because it only reflects transcriptional regulation.

We did, however, see a significant difference in the ratio of MMP-2/TIMP-1 gene expression in the ascending aorta between patients with BAV and TAV who had a dilated ascending aorta ≥4.0 cm. It may be, therefore, that it is the imbalance of these particular enzymes that, in part, leads to the increased diameter of the ascending aorta. Haemodynamic factors associated with the different types and degree of AV disease, may affect the local transcription and translation of these MMPs and TIMPs triggering an ongoing process of ECM degradation and dilatation of the vessel.

MMP-3, MMP-8, MMP-12 and TIMP-3 have significantly increased expression in AAA [10–13]. We did not study these MMPs and TIMPs and it may be that they have a more predominant role in ascending aorta dilatation.

Previous studies have shown that patients with BAV, independent of the functional state of the valve, have an increased diameter at every level of the aortic root and ascending aorta compared to patients with TAV [14]. In our study, these diameters were not significantly different comparing patients with BAV and TAV, but marginally smaller at the level of sinus of Valsalva for TAV.

In conclusion, we did not find any difference in the gene expression of the MMPs and TIMPs studied in the ascending aorta between patients with BAV and TAV, who had normal size ascending aorta. It may be that the genetic make-up of patients with BAV developing aneurysms of ascending aorta plays a lesser role than haemodynamic factors. A difference was observed in the expression of MMPs and TIMPs, particularly the ratio of MMP-2/TIMP-1, in the ascending aorta, between these two groups in the subset of patients with preoperatively dilated ascending aorta (diameter ≥4.0 cm).

4.1. Limitations of the study

The gene expression level of MMPs and TIMPs may not represent a complete picture of the abundance of the protein within the ascending aortic wall. Semiquantative morphometric analysis of immunohistochemical staining of the ascending aortic tissue would assess the relative abundance of the specific proteins. This would determine whether the level of gene expression correlates with the protein level in the tissue. We carried out immunohistochemical analysis to determine the protein levels of MMPs and TIMPs in a subset of ten patients. We found no significant correlation between gene expression and protein levels. To carry out immunohistochemistry for all patients would have entailed analysing approximately 4000 slides, which was not the primary aim of this study and out of its scope. Other MMPs, including MMP-3, -8 and -12 and TIMP-3, found to be upregulated in AAA formation may also have a role in ascending aortic dilatation and need further investigation.


   References
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
 References
 

  1. Freestone T, Turner RJ, Coady A, Higman DJ, Greenhalgh RM, Powell JT. Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol 1995; 15:1145–1151.[Abstract/Free Full Text]
  2. Shah PK. Inflammation, metalloproteinases, and increased proteolysis: an emerging pathophysiological paradigm in sortic aneurysm. Circulation 1997; 96:2115–2117.[Free Full Text]
  3. Wilson WRW, Anderton M, Schwalbe EC, Jones JL, Furness PN, Bell PRF, Thompson MM. Matrix metalloproteinase-8 and -9 are increased at the site of abdominal aortic aneurysm rupture. Circulation 2006; 113:438–445.[Abstract/Free Full Text]
  4. Keane MG, Wiegers SE, Plappert T, Pochettino A, Bavaria JE, Sutton MG. Bicuspid aortic valves are associated with aortic dilatation out of proportion to coexistent valvular lesions. Circulation 2000; 102:III35–39.[Medline]
  5. Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res 2002; 90:251–262.[Abstract/Free Full Text]
  6. Keeling WB, Armstrong PA, Stone PA, Bandyk DF, Shames ML. An overview of matrix metalloproteinases in the pathogenesis and treatment of abdominal aortic aneurysms 10.1177/153857440503900601. Vasc Endovascular Surg 2005; 39:457–464.[Abstract/Free Full Text]
  7. Goodall S, Crowther M, Hemingway DM, Bell PR, Thompson MM. Ubiquitous elevation of matrix metalloproteinase-2 expression in the vasculature of patients with abdominal aneurysms. Circulation 2001; 104:304–309.[Abstract/Free Full Text]
  8. Boyum J, Fellinger EK, Schmoker JD, Trombley L, McPartland K, Ittleman FP, Howard AB. Matrix metalloproteinase activity in thoracic aortic aneurysms associated with bicuspid and tricuspid aortic valves. J Thorac Cardiovasc Surg 2004; 127:686–691.[Abstract/Free Full Text]
  9. Fedak PW, de Sa MP, Verma S, Nili N, Kazemian P, Butany J, Strauss BH, Weisel RD, David TE. Vascular matrix remodeling in patients with bicuspid aortic valve malformations: implications for aortic dilatation. J Thorac Cardiovasc Surg 2003; 126:797–806.[Abstract/Free Full Text]
  10. Carrell TWG, Burnand KG, Wells GMA, Clements JM, Smith A. Stromelysin-1 (Matrix Metalloproteinase-3) and tissue inhibitor of metalloproteinase-3 are overexpressed in the wall of abdominal aortic aneurysms 10.1161/hc0402.102621. Circulation 2002; 105:477–482.[Abstract/Free Full Text]
  11. Higashikata T, Yamagishi M, Sasaki H, Minatoya K, Ogino H, Ishibashi-Ueda H, Hao H, Nagaya N, Tomoike H, Sakamoto A. Application of real-time RT-PCR to quantifying gene expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human abdominal aortic aneurysm. Atherosclerosis 2004; 177:353–360.[CrossRef][Medline]
  12. Wilson WRW, Schwalbe EC, Jones JL, Bell PRF, Thompson MM. Matrix metalloproteinase 8 (neutrophil collagenase) in the pathogenesis of abdominal aortic aneurysm. Br J Surg 2005; 92:828–833.[CrossRef][Medline]
  13. Longo GM, Buda SJ, Fiotta N, Xiong W, Griener T, Shapiro S, Baxter BT. MMP-12 has a role in abdominal aortic aneurysms in mice. Surgery 2005; 137:457–462.[CrossRef][Medline]
  14. Cecconi M, Manfrin M, Moraca A, Zanoli R, Colonna PL, Bettuzzi MG, Moretti S, Gabrielli D, Perna GP. Aortic dimensions in patients with bicuspid aortic valve without significant valve dysfunction. Am J Cardiol 2005; 95:292–294.[CrossRef][Medline]

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