DEPARTMENT OF PATHOLOGICAL ANATOMY
FACULTY OF MEDICINE
MASTER’S THESIS
Morphogenesis of post-stenotic aortic dilatation: Expression
of extracellular matrix metalloproteinase inducer (EMMPRIN)
in aortic media
Zeynab Ali Hussein
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TABLE OF CONTENT
TABLE OF CONTENT ... 2
SUMMARY ... 3
CLEARANCE ISSUES BY ETHIC COMMITTEE ... 5
ABREVIATIONS ... 6
CONFLICT OF INTEREST ... 7
INTRODUCTION ... 8
AIM AND OBJECTIVES ... 9
LITTERATURE REVIEW ... 10
Anatomy of aorta ... 10
Thoracic aorta ... 10
Abdominal aorta ... 11
Histology of aorta ... 12
Aetiology and pathophysiology of ascending aortic aneurysms ... 13
Post-stenotic aortic dilatation ... 15
Extracellular matrix metalloproteinase inducer (EMMPRIN) ... 16
Overview of metalloproteases ... 17
Structure and function of MMP ... 18
Tissue inhibitor of Matrix metalloproteases ... 19
Blood flow ... 20
Types of blood flow ... 21
Effects of hemodynamic changes on vascular smooth muscle cells ... 23
MATERIAL AND METHODS ... 24
Study subjects ... 24
Histology and immunohistochemistry ... 25
RESULTS ... 26
DISCUSSION ... 28
CONCLUSION ... 30
3
SUMMARY
Title: Morphogenesis of post-stenotic aortic dilatation; Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) in aortic media.
Aim: To evaluate the impact of the enzyme extracellular matrix metalloproteinases inducer (EMMRPIN) in morphogenesis of aortic dilatation due do aortic valve stenosis.
Objectives: 1) To evaluate expression of EMMPRIN in aortic samples from patients with aortic dilatation due to aortic valve stenosis. 2) To evaluate expression of EMMPRIN in aortic samples from patients without dilatative pathology. 3) To compare expression of
EMMPRIN in aortic samples with dilatation due to aortic valve stenosis and without dilatative pathology.
Methodology: To achieve the objectives, the following methods were used:
1) Study samples: histological slides with aortic specimens of the aortic wall taken from patients (n=13) with DPAA during surgery reconstruction of aortic dilatation due to aortic valve stenosis.
2) Reference samples: histological slides with aortic samples (punches) taken during CABG. The control group (n=14) underwent preoperative two-dimensional transthoracic
echocardiography to exclude dilatation of ascending aorta and valvar dysfunction.
Serial cross-sections (3 µm) thick of aortic tissue sections were deparaffinised and rehydrated by slide strainer Varistain Gemini (ThermoShendon). The sections were later counterstained with Mayer’s haematoxylin (J. T. Baker) and mounted using xylene-based mounting medium Consul-Mount TM (Shandon). For the evaluation of expression of EMMPRIN in the ascending aortic media, a semi-quantitative scoring method was used.
Research results: The median score of expression of EMMPRIN in the study group with dilatative pathology due to post-stenotic dilatation was 3 (range 0-6), while the median score of expression of EMMPRIN in the reference group without dilatative pathology was 1 (range 0-4). There was a significant higher expression of EMMPRIN in the DPAA study group compared to the reference group (p=0.0079).
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Conclusion: We found that there is a higher expression of EMMPRIN in aortic samples with dilatative pathology due to aortic valve stenosis compared to those aortic samples without dilatative pathology (median 3, range 0-6 vs median 1, range 0-4 (p=0.0079).
5
CLEARANCE ISSUES BY ETHIC COMMITTEE
Approved by: Kaunas Regional Biomedical Research Ethics Committee, Kaunas, Lithuania Biomedical research name:
Number: BEC-MF-140 Date: 2016.12.06
6
ABREVIATIONS
LUHS – Lithuanian University of Health Sciences
EMMPRIN - Extracellular matrix metalloproteinase inducer AS - Aortic valve stenosis
CABG - Coronary artery bypass grafting MMP – Matrix metalloproteinases
VSMC – Vascular smooth muscle cell SMC – smooth muscle cell
TGF-b1 – Transforming growth factor
DPAA – Dilatative pathology of ascending aorta
BAV – Bicuspid aortic valve
TAV – Tricuspid aortic valve
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CONFLICTS OF INTEREST
8
INTRODUCTION
Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as CD147, is a cell mebrane glycoprotein that stimulates the production of several matrix
metalloproteinases (MMPs) for tissue remodeling. EMMPRIN was initially identified in tumor cells where it was enriched and it’s role in cancer progression through MMPs initiation was accentuated.[1]
The degradation of the extracellular matrix (ECM) has been considered as the major function of matrix metalloproteinases (MMPs), this to permit normal tissue remodeling. Under normal physiological conditions, MMPs are expressed only when needed but their abnormal expression is most often associated with pathological conditions such as cancer, rheumatoid arthritis, fibrosis. [1]
EMMPRIN, other than being associated with oncological conditions, has also been reported to induce and activate the expression of MMPs in atherosclerotic plaque as well as it may be expressed in human aortic aneurysms and play a role in ECM remodeling, thereby the pathogenesis of aortic aneurysmal diseases. [2]
Aortic aneurysm is defined as localized weakened and malformed arterial wall architecture. It is a major disease processes and is becoming a common cause of death due to rupture or dissection. It can be both congenital or acquired and are generally classified according shape and size.[3]. The two most important disorders that predispose to aortic aneurysms are atherosclerosis and arterial hypertension; atherosclerosis is a greater risk factor in abdominal aortic aneurysm while arterial hypertension is a greater risk factor for ascending thoracic aorta.[4] They don’t share the same pathology but do share similar pathological phenotypes including remodelling of the aortic wall and decreased elastic fibres in the tunica media. [2]
As the medial layer of aorta degenerates, it leads to weakening of the aortic wall, which in turn results in aortic dilatation and formation of aneurysm. Normally, this can occur from normal aging process but can also be accelerated from one of the connective tissue syndromes e.g. Marfan’s disease or from the effects of chronic hypertension.[4]
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AIM AND OBJECTIVES
Aim: To evaluate the impact of the enzyme extracellular matrix metalloproteinases inducer (EMMRPIN) in morphogenesis of aortic dilatation due do aortic valve stenosis.
Objectives:
1) To evaluate expression of EMMPRIN in aortic samples from patients with aortic dilatation due to aortic valve stenosis.
2) To evaluate expression of EMMPRIN in aortic samples from patients without dilatative pathology.
3) To compare expression of EMMPRIN in aortic samples with dilatation due to aortic valve stenosis and without dilatative pathology.
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LITTERATURE REVIEW
Anatomy of aorta
Aorta is the greatest vessel in the body, it receives oxygenated blood from the left ventricle of the heart and supplies the organs in the body via the systemic circulation[5], [6]. It is divided into two principal segments: thoracic and abdominal. The thoracic portion of the aorta is moreover divided into ascending aorta, aortic arch and descending aorta. [5]–[7]
Figure 1. Schematic picture of aorta [8]
Thoracic aorta Ascending aorta
The ascending aorta is about 5 cm long segment, it emerges from aortic annulus at the region of aortic root, also known as lower segment of ascending aorta, which is composed of the sinuses of Valsalva and Sino-tubular junction (STJ).[5] The tubular ascending segment, known as the upper part, begins at the STJ and ends just prior to the innominate
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front of the body and do not give off any branches if compared to the parts, the aortic arch and descending aorta. [5], [6]
Aortic arch
The aortic arch begins at the level of second intercostal joint on the right side of the body and is a continuation of the ascending aorta.[5], [6] As the name implies, it as an transverse arch that connects ascending aorta and descending aorta. [5]This short segment gives off three branches; the innominate(brachiocephalic) artery, left carotid artery and the left subclavian artery, that together supplies the head and the arms with oxygenated blood.[5]
First branch of the arch is also the largest branch, the brachiocephalic artery which supplies right division of the head and brain and the right arm. Second branch is the left common carotid artery which supplies the left portion of the brain. The last, third branch is the left subclavian artery that supplies the left arm.[5]
Descending aorta
The descending aorta is a continuation of the aortic arch and is located towards the back of the body. It starts with the last branch of the aortic arch, the left subclavian artery and ends at level of the celiac artery, which is the first branch of the abdominal aorta.[5], [6] Descending aorta gives off several arterial branches that supplies the bronchi, oesophagus, pericardium and are named according the organ they supply.[5], [6]
Abdominal aorta
Abdominal aorta is approximately 13 cm long, it begins as a continuation of thoracic aorta, at the level of T12 vertebrae and extends to the level of L4 where aorta ends by
bifurcating into right and left common iliac artery. The branches given off in abdominal part of aorta includes left and right renal artery, celiac artery, superior mesenteric artery and the inferior mesenteric artery. [5]
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Histology of aorta
The arterial vessel as the aorta are made up of three layers;
The outer layer: Tunica Adventitia, is made up by connective tissue and has a “vasa vasorum” since larger arteries needs their own blood supply.[9]–[11]
Middle layer: Tunica media, is made up of relatively few smooth muscle fibres, elastic fibres with concentric fenestrated sheets of elastin and collagen. [9], [10]
Inner layer, Tunica intima, is made up of an epithelium, a single layer of flattened endothelial cells. In this layer, there is also a layer of elastin that are in rich collagen and a layer of fibroblasts.[9], [10]
Figure 2.Histological picture of aorta[12]
The walls of aorta and larger arteries contains elastic tissue which mostly is found in the internal elastic lamina that is prominent band that separates the inner layer from the middle layer. During systolic activity of the heart, the vessels are stretched out by the force of cardiac ejection and during the diastole, the elastic tissues allows them to recoil. [7]
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The expansion of the aorta during systole allows kinetic energy from left ventricular contraction to be stored as potential energy in aortic wall and used during diastole instead. In diastole, the potential energy is transformed back into kinetic energy and causes the blood to move distally into the arterial bed. [7]
With aging, elastic fibres become fragmented and smooth muscle withdraws, this in combination leads to increased stiffness and weakening of the aortic wall which predisposes to dilatation of aorta.[7]
Aetiology and pathophysiology of ascending aortic aneurysms
The pathophysiology of aneurysm formation is far complex and involves
inflammation, proteolysis and disturbed function of smooth muscle cells in the aortic wall. It is a multifactorial pathology[7]
Most aneurysms result from loss of structural integrity and aortic wall strength. It is thought that a combination of breakdown of extracellular matrix proteins and mechanical factors cause medial degeneration. [4]
1. Marfans syndrome
In younger patients, cystic medial degeneration is classically associated with Marfans syndrome. This is a heritable autosomal-dominant disorder caused by mutations in one of the genes for fibrillin-1, a structural protein that is the major component of microfibrils of elastin. The outcomes of the mutations is both a decrease in the amount of elastin in the aortic wall and a loss of elastin’s normally organized structure. This overall results in abnormal elastic properties that lead to progressive increases in stiffness and dilatation.[7], [13], [14]
2. Familial Thoracic Aortic Aneurysm Syndrome
Familial thoracic aortic aneurysm and dissection (TAAD) is seen in patients who have thoracic aortic disease associated with a family history of aneurysmal disease. Familial
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TAAD includes patients with a dilated aorta or aneurysm affecting other vessels, and a family history of dissection, rupture, or sudden unexplained death.[15], [16]
3. Atherosclerosis
Atherosclerosis is more an infrequent cause of ascending thoracic aortic aneurysms but plays a role in ascending thoracic aortic aneurysm[4]
4. Turner Syndrome
Is a sex aneuploidy syndrome in which only a single X chromosome is present (45 XO). In this patient group, cardiovascular diseases are common including Bicuspid Aortic Valve, elongation of the transverse arch. Aortic dilatation typically involves the root of the ascending aorta and sometimes extending to the descending aorta. [15]
5. Aortitis
Aortitis is the pathological term for inflammation of the aortic wall where the most common causes are the large-vessel vasculitides giant cell arteritis (GCA) and Takayasu arteritis. It is characterized by presence of inflammation of the adventitia and media, often with giant cells (Figure 3C). The pathogenesis of both GCA and Takayasu arteritis remains unknown but it is thought to be antigen-driven cell-mediated autoimmune processes, although the specific antigenic stimuli have not been identified.
6. Bicuspid aortic valve
Bicuspid aortic valve (BAV) disease is the most common congenital heart disease. It is associated with valvular complications (aortic stenosis or regurgitation) as well as vascular complications such as ascending aorta dilatation. Up to 83% of patients with BAV will develop ascending aorta dilatation.
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Figure 3. Macroscopic (A) and Histological (B) view of Giant cell aortitis (C) Tricuspid aortic valve stenosis. B- staining by H+E, scale 100 microns[17]
Post-stenotic aortic dilatation
Tricuspid aortic valve stenosis (Fig. 3C) is the third most common valvular heart disease following coronary artery disease and hypertension, in Europe and North America. It
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is a chronic progressive disease that begins with thickening and calcification of the valve cusps and progresses into heavily calcified, stiff cusps that results in reduced leaflet motion and valve area, resulting in increased pressure gradients across the valve [18], [19]
Post-stenotic dilatation tends to occur in patients with AS and/or aortic regurgitation, in patients with a haemodynamically normal bicuspid aortic valve and in patients after aortic valve replacement.
Post-stenotic aortic dilatation is a dilatation of the vessel wall of the ascending aorta, >4.0 cm, distal to a stenotic/malformed aortic valve (AV). It is a progressive process where the vessel dilatation is usually > 0.3 cm/year. As this progression continues, it leads to aortic dissection and rupture, both of which are potentially fatal.[18], [19]
Extracellular matrix metalloproteinase inducer (EMMPRIN)
Extracellular matrix metalloproteinase inducer (EMMPRIN), known as Basigin (BSG) in mice and as Cluster of differentiation (CD147). It was primarily identified back in the 1990’s as a cell-surface glycoprotein which belongs to the immunoglobulin
superfamily.[20]
The well-known and studied function of EMMPRIN/CD147 is its expression on cancer cells to induce the production of different matrix metalloproteinases (MMPs) in cancer cells and fibroblasts following epithelial-stromal interaction leading to degradation of
extracellular matrix. Other than oncological pathology, EMMPRIN’s upregulation has been implicated in many other pathological processes such as rheumatoid arthritis, lung injury, chronic liver diseases, atherosclerosis and heart failure. [20]
EMMPRIN has two C2-like immunoglobulin extracellular domains; a
transmembrane domain and a short cytoplasmic domain. The region of extracellular domain contains three conserved N-glycosylation sites that are differently glycosylated and the process glycosylation has shown to determine stimulating activity of EMMPRIN. Different modes of glycosylation produces different forms of EMMPRIN and is associated with different MMP expression pattern.[21] (Figure 4)
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Figure 4. EMMPRIN signalling pathway[22]
Previously, it has been reported that EMMPRIN induces and activates the expression of MMP-9 in monocytes and MMP-2 in vascular smooth muscle cells in acute myocardial infarction, as well as it might be expressed in human aortic aneurysms and therefore play a role in the pathogenesis of aortic aneurysmal diseases.[21]
It has also been reported that the increased expression of EMMPRIN in aortic aneurysmal diseases is of vascular smooth muscle cells origin rather than
monocyte/macrophages origin.[21]
Overview of metalloproteases
Matrix metalloproteinases (MMP) are a group of enzymes that belong to the family of proteases and are responsible for collagen and proteoglycans degradations in extracellular matrix(ECM). ECM is mostly composed of the structural component collagen. It has an important role in maintaining the stability of organs and support their structural integrity.[14], [21], [23]
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There are currently 26 MMP’s described in human, they are divided into six functional groups:
Collagenases (MMP-1, MMP-8, MMP-13) which degrades interstitial collagens I, II and III at a specific site close to the substrate N-terminus.
Gelatinases (MMP-9, MMP-2) These enzymes degrade denatured collagens and are specific for the degradation of collagen types I, IV, V, VII, X, IX, elastin, fibronectin, aggrecan, vitronectin, laminin.
Stromelysins (MMP-3,MMP-7,MMP-10,MMP-13) which have a substrate activity capable of cleaving proteoglycans, collagens and fibronectin[14], [21], [23]
Matrilysins (MM-7 and -26) play an important role in the degradation of ECM proteins like type IV collagen, laminin and entactin
Membrane-type MMP (-14,-15,-16,-17,-24,-25) have shown to catalyse the activation of pro-gelatinase A and to degrade variety of ECM substrates.
Structure and function of MMP
The matrix metalloproteinases are initially secreted as inactive enzymes and can be activated either by other proteinases including other MMPs. They are synthesized by many different cell types including endothelial cells, smooth muscle cells, macrophages and fibroblasts. For activation of MMPs, the “cysteine switch” which blocks the catalytic site by a zinc/cysteine interaction, must be removed. Once this cysteine/zinc interaction is removed, a MMP intermediate is formed which later is fully activated by other MMP intermediates or fully active MMPs.[21], [23]
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Figure 5. Activation of MMP in three steps[24]
Firstly, secreted as Pro-MMP , then partially activated once the “Cysteine switch” is disrupted and finally fully activated by other MMPs and removal of the pro-peptide region. [24]
The production of MMPs is regulated by levels of MMP mRNA which in turn is affected by several important factors including hormones, cytokines, growth factors, and hypoxia. MMPs activation is regulated by the levels and activity of MMPs, other proteinases and endogenous tissue inhibitor of metalloproteinases (TIMPs). [21], [24]
Tissue inhibitor of Matrix metalloproteases
MMPs act on the cell surface or in the extracellular space and the activity of MMPs are controlled by endogenous inhibitors and the tissue inhibitor of metalloproteases.
TIMPs are a family of four protease inhibitors: TIMP-1, TIMP-2, TIMP-3 and TIMP-4, they show to some degree target specificity for MMP’s e.g. TIMP-2 has preference for MMP2. [21][25]
TIMPS are all expressed in the heart and vascular wall, their expression can be regulated by different stimuli including angiotensin II and inflammatory cytokines.
Degradation of extracellular matrix in normal and pathological states are regulated by the balance between TIMPS and MMPs. [25]
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Since they can regulate MMP activity, they also play a major role in tissue remodelling. TIMPS have shown that when it is dysregulated, it becomes a cause of many disease including cardiovascular disorder such as aneurysm.[21][25]
Blood flow
There are two types of mechanical forces which normal blood vessels are exposed to (a) circumferential stretch acting tangentially on the wall of a blood vessel and is directly related to pressure and dimensions of the vessel and (b) shear stress that acting longitudinally, at the blood/endothelium interface and is related to the velocity of flow. [19], [24]
Blood pressure is defined as the force that the circulating blood exerts on the walls of arteries and creates a strain on the vessel wall. The forces a normal blood vessels are exposed to, are counterbalanced by intraparietal tangential forces in longitudinal and circumferential directions exerted by different elements of the vessel wall. On the other hand, all layers of the arterial wall are exposed to circumferential tension but in different degrees. [19], [24]
The tension per unit length is described by Laplace’s Law: T=Pr/h where, T is the wall tension, P is the blood pressure, r is the vessel radius and h is the thickness of the wall.
The blood flow in our circulatory system exerts a frictional force on the luminal surface of endothelium. The frictional force is referred to as shear stress and is defined as bloods viscosity and velocity.[24][19]
There is a well-established relationship between circumferential stress and the structure of the vessels wall. It is known that increased arterial pressure are both associated with increase in extracellular matrix (ECM) production and remodelling of the smooth muscle cells.[24]
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Figure 6. Shear stress and cyclic strain[24]
A) Laminar shear stress within an artery where frictional forces between blood and the vessel wall causes fast blood velocity at the centre of the vessel and reduction at the wall of the vessel.[24]
B) Blood pressure results in dilation, a circumferential stretch of the vessel wall in a direction perpendicular to the direction of blood flow. [24]
Types of blood flow
Laminar type of blood flow within a vessel is when the blood is flowing at a steady rate in streamlines, with each layer of blood remaining in the same distance from the vessel wall and the velocity in the centre of the vessel is far greater than that toward outer
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The laminar flow is described by the equation 𝜏 = 4𝜇𝑄/ π 𝑟3 where 𝜏 is shear stress, 𝜇 is blood viscosity, Q is flow rate and r is the vessel radius.[19], [24]
Turbulent type of blood flow is the opposite to laminar flow in which blood is flowing crosswise in all directions within the vessel, forming whorls rather than
streamlined.[26]
The tendency for turbulent flow increases in direct proportion to the velocity of blood flow, diameter of blood vessel, density of the blood and its viscosity. Turbulent blood flow occurs when the rate of flow becomes too great, this occurs under some circumstances, e.g. when it passes by an obstruction in a vessel, passes a rough surface or makes a sharp turn.(Fig.10)[26]
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Effects of hemodynamic changes on vascular smooth muscle cells
As explained earlier, increased blood pressure enforces increased mechanical stress on the vascular wall, and mechanical strain is a mitogenic stimulus for vascular smooth muscle (VSM) cells. The vascular wall of increased pressure is constantly modified because there is progressive accumulation of extracellular matrix (ECM) that is produced by the smooth muscle cells within the vascular wall. Several factors have been implicated in
cardiovascular matrix accumulation such as neurohumoral factors and the vascular wall stress as consequence from the increase in blood pressure.[28]
The arterial wall is exposed to mechanical strain during the cardiac cycle and of which is mostly experienced by the vascular smooth muscle cells This mechanical strain in turn has shown to influence VSM cell growth, phenotype and function. The accumulation of extracellular matrix in the vascular wall do not only affect the cross-sectional area available to flow but it also affects the arterial wall stiffness and thereby arterial compliance, pulse wave propagation and pulse pressure.[28]
A previous study by O’Callaghan and Williams suggest that TGF- β1, that is a potent fibro genic cytokine, plays a key role in the regulation of mechanical strain–induced matrix synthesis by human vascular smooth muscle cells.[28]
They examined whether human VSM cells have the potential to produce TGF-β1 by examined the expression of TGF-β1 mRNA. This was done by using human VSM cells that were rendered quiescent by serum depletion for 48 hours before being exposed to mechanical strain for up to 6 hours and measured the expression of TGF-β1. [28]
The presented results followed rapidly increased TGF-β1 mRNA expression during mechanical strain compared to baseline and this also explains that human VSM cells express TGF-b1 mRNA and also that chronic cyclical mechanical strain is a powerful stimulus for TGF-b1 mRNA expression by these human cells[28]
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MATERIAL AND METHODS Study subjects
The study was carried out at the Laboratory of Molecular Cardiology, Institute of Cardiology of the Lithuanian University of Health Sciences (LUHS), Kaunas.
The study enrolled 13 patients in total (10 males, 3 females) with the median age of 61 years (range 43-76 years). The samples studied were composed of aortic tissue taken from patients undergoing aortic reconstruction due to DPAA at the Department of Cardiac,
Thoracic and Vascular surgery.
The aortic samples were collected by researchers of Laboratory of Molecular Cardiology in collaboration with cardio surgeons from Department of Cardiac, Thoracic and Vascular Surgery of the Lithuanian University of Health Sciences (LUHS) in 2004-2014.
Aortic wall specimens, punches biopsies, that were taken from the ascending aorta, at the site of proximal bypass anastomosis during coronary artery bypass grafting (CABG) surgery, were used as control group. The reference group enrolled 14 patients (11 males, 3 females) with the median age of 65,5 years (range 56-85 years), who underwent preoperative two-dimensional transthoracic echocardiography to exclude dilatation of the ascending aorta and valvar dysfunction. All the data were collected prospectively and analysed
retrospectively.
Information on (1) hypertension (systolic blood pressure ≥ 140 mm Hg and/or diastolic blood pressure ≥ 90 mm Hg); (2) assessment of history of diabetes mellitus was taken from the patients collected health status data.
The specimens of aortic wall samples taken during aortic reconstruction surgery were fixed in a 10 % neutral buffered formalin for 24 hours before being processed for routine paraffin embedding in the Laboratory of Cardiac Pathology of Institute of Cardiology, LUHS.
The present study was approved by the Ethics Committee of Lithuanian University of Health Sciences. All the patients were informed about the study and gave their consent to participate.
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Histology and immunohistochemistry
Serial cross-sections (3 µm) thick of aortic tissue sections were deparaffinised and rehydrated by slide strainer Varistain Gemini (ThermoShendon). The sections were washed with distilled water and heated in Tris/EDTA buffer (pH 9.0) for 8 min at 110°C in Microwave Histoprocessor RHS-1 (Milestone, Microwave Laboratory Systems). Shandon Coverplate system was used for immunohistochemically labelling. After blocking the activity of endogenous peroxidases, all slides were incubated in the primary antibody buffer for 1 hour at a dilution of 1:25 for EMMPRIN (CD147) (Novocastra, Great Britain) in an antibody diluent (DakoCytomation), followed by sequential 30-min incubations with Advanced TM HRP Link and Advanced TM HRP Enzyme (DakoCytomation). The binding of antibodies was detected by Liquid DAB + Substrate-Chromogen System (DakoCytomation). Finally, the sections were counterstained with Mayer’s haematoxylin (J. T. Baker) and mounted using xylene-based mounting medium Consul-Mount TM (Shandon). Dermal tissue was used as positive control. Negative control - nonspecific negative (using IgG) reagents control.
For the evaluation of expression of EMMPRIN in the ascending aortic media, a semi-quantitative scoring method was used.
Figure 8. Expression of EMMPRIN in aortic media (score 3). Arrow indicates smooth muscle cell. Scale 50 microns
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RESULTS
The demographic and clinical demographics of DPAA patients and the reference group are reported in Table 1. These data did not differ according age, gender, hypertension and diabetes between the groups studied. The study group included sub-groups of cases with both bicuspid (6 BAV) and tricuspid aortic valve (7 TAV) while in the reference group were only cases with TAV.
Table 1. Demographic and Clinical characteristics of study participants
Characteristics DPAA patients
(n = 13)
Reference group
(n = 14)
P value
Age, years, median (range) 61 (43-76) 67.5 (56-85) 0.459
Males (%) 10 (76.9) 11 (78.6) 0.918
Hypertension (%) 9 (69.2) 12 (85.7) 0.303
Diabetes mellitus (%) 1 (7.7) 1 (7.1) 0.956
Bicuspid valve (%) 6 (46.2) 0 (0) 0.0058
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Table 2. Score of expression of EMMPRIN
Expression of EMMPRIN DPAA patients
(n = 13)
Reference group
(n = 14)
P value
Median (range) score 3 (0-6) 1 (0-4) 0.0079
EMMPRIN: Extracellular matrix metalloproteinase inducer, DPAA: Dilatative pathology of ascending aorta.
The expression of EMMPRINN was significantly higher (P<0.0079) in the aortic wall from patients with DPAA when compared with control specimens. (Table 2)
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When evaluating the expression of EMPRINN, TAV in the reference group was compared to TAV respectively BAV in the DPAA group as well as TAV compared to BAV in the DPAA group. (Figure 9)
The results presented shows that expression of EMMPRIN is significantly higher (P=0.003) in TAV patients of DPAA group compared to the control group. There was no difference between the TAV patients in control group compared to BAV patients in the DPAA group (P=0.197). Within the DPAA group, the expression of EMMPRIN of TAV compared to BAV showed a tendency to be higher, but it did not reach the significant level (median score 4, range 1-6 respectively median score 1.5, range 0-4, P=0.082).
DISCUSSION
Vascular smooth muscle cells (SMCs) are the main source of ECM proteins in the aortic media. The interaction between SMCs and ECM proteins are crucial for the structural and functional integrity of the aortic wall. At homeostasis, the mature vascular SMCs sustain a quiescent differentiated state to perform contractile function. This phenotype is designated as contractile or as differentiated phenotype. [29], [30]
In vascular pathologies, such as atherosclerosis or in mechanical injuries, the
vascular SMCs can differentiate from the contractile to a so-called synthetic type and produce various substances involved in vascular remodelling, including ECM proteins, growth factors, and proteases.[29], [30]
Overexpression of MMP in the aortic wall is believed to play an important role in the pathogenesis of dilative aortic pathologies, specially MMP-1, -2, and -9 that belongs to
gelatinases group of MMPs. MMP-1 (interstitial collagenase) degrades type I, II, and III collagens, while MMP-2that is primarily produced by SMCs degrades type IV collagen and elastin.[2], [29], [30]
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It has been reported that EMMPRIN may have a key regulatory role in MMP activity in cardiovascular pathology. The well-known and studied function of EMMPRIN/CD147 is its expression on cancer cells to induce the production of different matrix metalloproteinases (MMPs) in cancer cells and fibroblasts following epithelial-stromal interaction, leading to degradation of extracellular matrix. Other than oncological pathology, EMMPRIN’s upregulation has been implicated in many other pathological processes such as rheumatoid arthritis, lung injury, chronic liver diseases, atherosclerosis and heart failure. [20]
A study that was carried out by Xiao-Feng CHEN[2], showed for the first time the presence and the increased expression of EMMPRIN in human thoracic or abdominal
aneurysmal aortas and that EMMPRIN was locally expressed mainly by vascular SMCs in the aneurysmal lesion.[2]
There are no previous studies reported on the morphogenesis or the expression of EMMPRIN in post-stenotic aortic dilatation. We have for the first time showed that the expression of EMPRINN is present in vascular SMCs of aorta and is upregulated in patients with post-stenotic aortic dilatation when comparing to those without DPAA.
From the results of the present study and previous studies about both MMP’s and EMMPRIN, that it can alter the expression and activity of MMP’s in different cell types including SMC’s, it was expected that the expression of EMMPRIN would be up-regulated in the study group of post-stenotic aortic dilatation. Even though the exact mechanism of how EMMPRIN may affect MMP activation in aortic aneurysmal diseases remains unclear, we think that the increased expression of EMMPRIN in the aneurysmal wall might affect the MMP activity and the organization of collagen fibrils and elastic fibres. [2], [21][29]
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CONCLUSIONS
1. Median score of expression of EMMPRIN in aortic samples taken from patients operated due to aortic dilatation caused by aortic valve stenosis was 3 (range 0-6). Within the study group, the expression of EMMPRIN in aortic samples taken from patient with tricuspid aortic valves showed a tendency to be higher than in samples from patients with bicuspid aortic valve, but it did not reach the significant level (median score 4, range 1-6, and median 1.5, range 0-4, P=0.082, respectively)
2. Median score of of EMMPRIN in aortic samples taken from patients without ascending aorta dilatation was 1 (range 0-4).
3. Expression of EMMPRIN in aortic samples taken from patients operated due to aortic dilatation caused by aortic valve stenosis was significantly higher than in aortic samples taken from patients without ascending aorta dilatation ( median score 3, range 0-6, and median score 1, range 0-4, P=0.0079, respectively).
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