Abstract
Background. The study evaluated macrophage cytokines and macrophage metalloprotease (MMP)-12 levels in patients with Stanford-A acute aortic dissection (AAD) and in patients with critical carotid artery stenosis (CAS) compared with patients matched for age, sex, and traditional cardiovascular risk factors (RF). The aim was to identify possible early serum markers of risk for atherosclerotic complications.
Materials and methods. We selected 65 patients: 23 AAD patients, 21 CAS patients, 21 RF, and 10 healthy subjects (HS). In each patient and control serum, levels of interleukin (IL)-6, IL-8, tumour necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, vascular endothelial growth factor (VEGF), and MMP-12 were assessed by ELISA.
Results. A significant increase of MMP-12, IL-6, and IL-8 levels in AAD versus CAS was found. Moreover, MMP-12 was shown to be significantly higher in AAD versus RF, but not in CAS versus RF. A significant increase of IL-6, IL-8, MCP-1, TNF-α, and VEGF levels was observed both in AAD and CAS versus RF.
Conclusions. The results suggest that MMP-12 may be considered to be a specific marker of Stanford-A AAD. Furthermore, the study confirmed that in AAD and CAS macrophage cytokines play a key role in the progression of the atherosclerotic disease towards complications.
Key messages
Metalloprotease (MMP)-12 can be considered a specific marker of Stanford-A acute aortic dissection.
Macrophage activation represents a common denominator in the progression of the atherosclerotic disease either occlusive or breaking.
IL-6 and IL-8 seem to be specifically involved in the process underlying aortic dissection.
Introduction
It is well known that atherosclerosis is an inflammatory disease, in which the complex interaction between immune cells and inflammatory mediators drives the growth of atherosclerotic lesions and their progression towards complications (Citation1). Adhesion molecules, pro-inflammatory cytokines, chemokines, hydrolytic enzymes, and growth factors are all involved in this inflammatory process (Citation2). Recently, it has been demonstrated that intra-parietal inflammation can also lead to aneurisms and dissections at any point of the aorta, including the ascending tract (Citation3). Thus, athero-occlusive disease (AOD) and acute aortic dissection (AAD) seem to represent different faces of the same atherosclerotic pathology. Despite sharing the same risk factors and intra-parietal inflammation, these diseases differ greatly in their immunological pattern (Citation2,Citation3). Atherosclerotic plaque is associated with lymphocyte T CD4+ (T-helper 1) response, which would drive the atherosclerotic process towards a proliferative pattern (Citation4), whereas macrophages seem to be related to the lytic pattern involved both in plaque destabilization (Citation2–5) and in arterial wall weakening causing AAD (Citation6). On the basis of such premises, we decided to investigate macrophage products in patients with Stanford-A AAD compared with patients with asymptomatic critical carotid artery stenosis. The aim was to determine whether a suitable marker of AAD can be identified among macrophage products.
Materials and methods
Study population
From January 2012 to December 2012, a total of 65 patients were selected. Of these, 23 were undergoing type A Stanford AAD surgical repair at the Attilio Reale Heart and Great Vessels Department, ‘Sapienza’ University of Rome (AAD group). All the patients had 1) type A Stanford AAD; 2) no history of neoplasm or autoimmune, infectious, or inflammatory systemic disease; 3) no presence of genetic syndromes known to be responsible for aortic disease; and 4) no family history of aortic dissection or aneurysm. Twenty-one patients had asymptomatic critical carotid artery stenosis (CAS group), which according to the international literature means patients with no acute cerebrovascular disease (stroke or TIA) (Citation7,Citation8), and were candidates for thromboendoarterectomy in the Vascular Surgery Department, Ospedale Sant’Andrea, ‘Sapienza’ University of Rome. All patients had 1) no history of neoplasm, autoimmune, infectious, or inflammatory systemic disease; and 2) no cardiovascular disease. Finally, 21 patients matched for age, sex, and traditional cardiovascular risk factors (RF group), attending the Outpatient Department of Atherosclerosis and Dyslipidaemia, Ospedale Sant’Andrea, ‘Sapienza’ University of Rome were selected as the controls. All the patients underwent carotid ultrasound and were under pharmacological treatment; they were considered under control if they had glycosylated haemoglobin levels < 6.5% (Citation9), LDL values < 160 mg/dL (Citation10), and arterial blood pressure < 140/90 mmHg (Citation11). Patients were excluded from the control group if they had 1) genetic syndromes known to cause aortic disease; 2) family history of aortic dissection/aneurysm or cardiovascular disease; 3) history of aortic dissection/aneurysm or cardiovascular disease; 4) critical carotid artery stenosis (Citation7,Citation8). Among those attending the Outpatient Department of Internal Medicine, Ospedale Sant’Andrea, ‘Sapienza’ University of Rome, 10 healthy subjects (HS group) matched for age and sex were further selected as controls on the basis of the following exclusion criteria: 1) no presence of genetic syndromes known to cause aortic disease; 2) no family history of aortic dissection/aneurysm or cardiovascular disease; 3) no history of aortic dissection/aneurysm or cardiovascular disease; 4) no smoking habit; 5) no diabetes mellitus (Citation9); 6) no dyslipidaemia (Citation10); and 7) no uncontrolled arterial hypertension (Citation11).
A venous blood sample was taken from each patient, just prior to surgery, and from each control to evaluate serum levels of interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and metalloprotease (MMP)-12.
The study was performed according to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Faculty of Medicine. Written informed consent was obtained from each control and patient or from an authorized family member.
Serum IL-6, IL-8, MCP-1, VEGF, TNF-α, and MMP-12 levels
Serum IL-6, IL-8, MCP-1, VEGF, TNF-α, and MMP-12 levels were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (IL-6, IL-8, and VEGF-A gen-probe Diaclone SAS; MCP-1 IBL International GMBH; MMP-12 Cusabio Biotech Co, Ltd) according to the manufacturer's instructions. Briefly, after two washes of the plates with 400 μL/well of wash buffer, 100 μL/well of sample diluent were added to standard wells, and then cytokine standards were progressively diluted; 100 μL/well of sample were added to the wells. The plates were incubated at room temperature (18–25°C) on a microplate shaker (100 rpm) from 1 to 3 h according to the molecule under evaluation. Then the plates were washed three times with 300 μL/well of wash buffer, and 100 μL/well of biotin-conjugate antibody were added. After 1 h of incubation at room temperature (18–25°C) on a microplate shaker (100 rpm), the plates were washed six times with 400 μL/well of wash buffer, and 100 μL/well of streptavidin-HRP were added. Following 1 h of incubation at room temperature (18–25°C) on a microplate shaker (100 rpm), the plates were washed six times with 400 μL/well of wash buffer; then 100 μL/well of TMB substrate solution were added for 30 min at room temperature (18–25°C) in the dark. Stop solution was added, 100 μL/well, and optical density (OD) was read for each well with a microplate reader set to 450 nm. The average absorbance values were evaluated in duplicate for each set of standards and samples. To determine the concentration of circulating IL-6, IL-8, MCP-1, VEGF, TNF-α, and MMP-12, a standard curve was created. IL-6, IL-8, MCP-1, VEGF, and TNF-α concentrations were expressed as pg/mL; MMP-12 as ng/mL.
Statistical analysis
The results were expressed as median and mean ± standard deviation (SD). The non-parametric Mann–Whitney test was used to perform the statistical analysis; a P value < 0.05 was considered significant. All the statistical procedures were performed by GraphPad Prism 4 software (GraphPad Software, Inc.)
Results
Demographics and baseline characteristics
Demographic and baseline characteristics of the populations under evaluation are reported in .
Table I. Demographic and baseline characteristics of the populations.
No significant difference was observed regarding age, sex, and traditional cardiovascular risk factors among the AAD, RF, and CAS groups, and no significant difference was found between each group and HS regarding age and sex.
AAD versus HS and CAS versus HS
In AAD the median values of MMP-12, IL-6, IL-8, MCP-1, TNF-α, and VEGF were 19.30 ng/mL, 162.10 pg/mL, 98.93 pg/mL, 402 pg/mL, 7.04 pg/mL, and 176.30 pg/mL, respectively ().
Table II. Serum MMP-12, IL-6, IL-8, MCP-1, TNF-α, and VEGF levels.
In CAS the median values of MMP-12, IL-6, IL-8, MCP-1, TNF-α, and VEGF were 8.80 ng/mL, 86 pg/mL, 45.60 pg/mL, 387.9 pg/mL, 9.12 pg/mL, and 205.50 pg/mL, respectively ().
In HS the median values of MMP-12, IL-6, IL-8, MCP-1, TNF-α, and VEGF were 2.70 ng/mL, 0.70 pg/mL, 4.75 pg/mL, 132.80 pg/mL, 2.73 pg/mL, and 26.99 pg/mL, respectively ().
A significant increase in the serum levels of MMP-12, IL-6, IL-8, MCP-1, TNF-α, and VEGF was observed in AAD versus HS (P values 0.0006, < 0.0001, < 0.0001, < 0.0001, 0.0003, and 0.0138, respectively) () and in CAS versus HS (P values 0.0013, < 0.0001, < 0.0001, < 0.0001, 0.0167, and 0.0073, respectively) ().
Table III. Statistical analysis: P values from Mann–Whitney non-parametric test.
AAD versus RF and CAS versus RF
In RF the median values of MMP-12, IL-6, IL-8, MCP-1, TNF-α, and VEGF were 6.25 ng/mL, 3.36 pg/mL, 4.41 pg/mL, 245.10 pg/mL, 2.36 pg/mL, and 43.75 pg/mL, respectively ().
A significant increase in MMP-12 (P = 0.0017), IL-6 (P < 0.0001), IL-8 (P < 0.0001), MCP-1 (P = 0.0002), TNF-α (P = 0.0003), and VEGF (P = 0.0315) serum levels was observed in AAD versus RF (). Moreover a significant increase of IL-6 (P < 0.0001), IL-8 (P < 0.0001), MCP-1 (P = 0.004), TNF-α (P = 0.0091), and VEGF (P = 0.0167) was found in CAS versus RF, whereas MMP-12 (P = 0.1832) levels were not significantly higher in CAS versus RF ().
AAD versus CAS
In AAD versus CAS a significant increase in MMP-12 (P = 0.0164), IL-6 (P = 0.0364), and IL-8 (P = 0.0136) serum levels was observed, whereas no significant difference was observed in MCP-1 (P = 0.6905), TNF-α (P = 0.8852), and VEGF (P = 0.8546) values ().
Serum levels of each molecule under evaluation are reported in and the P values in .
Discussion
The results confirm the major role of macrophage cytokines in the progression and complications of atherosclerotic disease both in its occlusive and in its breaking form. In fact, patients were selected showing the opposite clinical extreme of the same atherosclerotic pathology, and a significant increase was found in serum levels of IL-6, IL-8, MCP-1, TNF-α, VEGF, and MMP-12 both in AAD and in CAS versus HS, but also of IL-6, IL-8, MCP-1, TNF-α, and VEGF in AAD and CAS versus RF. This suggests that macrophage activation is strongly related to the clinical outcome of the atherosclerotic disease. In particular, the significant differences observed versus RF support the hypothesis that patients with a high risk of atherosclerotic progression and complications, either athero-occlusive or dilatative, display high serum macrophage cytokine levels. Macrophage activation, therefore, seems to be responsible for the establishment of a lytic state which leads on to plaque growth, on the one hand, and, on the other, to aortic wall rupture.
The result obtained from the comparison of MMP-12 levels seems to be of particular interest, since it strongly suggests that MMP-12 may be considered a new marker of Stanford-A AAD in patients with traditional cardiovascular risk factors with no genetic predisposition. Indeed, the study found that MMP-12 levels were significantly increased in AAD patients versus CAS and versus all control groups, whereas its values did not significantly differ in CAS versus RF, suggesting that MMP-12 is specifically released in patients at high risk of dissection. These results are in agreement with the literature, where it has been demonstrated that MMP-12, a matrix-degrading elastase, exclusively released by macrophages (Citation12), has a specific role in the pathogenesis of aneurysms occurring both in ascending and abdominal aorta (Citation12–14). This specificity makes MMP-12 a very interesting matrix-degrading protein. It is well known that an imbalance between MMP-1, -2, and -9 levels and their specific tissue inhibitors leads to matrix remodelling both in athero-occlusive diseases (Citation15) and in AAD (Citation16,Citation17). These MMPs, therefore, are of little use as markers of one of these two conditions, whereas MMP-12, as also demonstrated in our results, seems to represent a useful marker to identify, among symptomatic patients, those at higher risk of dissection. Patients with Stanford-A AAD, indeed, often display symptoms and signs of other cerebrovascular and cardiovascular pathologies, such as TIA, stroke, or acute myocardial infarction.
Our results also showed a significant increase in IL-6 and IL-8 levels in AAD versus CAS. These data are in agreement with Lindeman et al., who have demonstrated that IL-6 and IL-8 hyperexpression is associated with abdominal aortic aneurysm (AAA) growth and may allow early distinction of AAA from athero-occlusive lesions (Citation18). Although IL-6 and IL-8 release occurs early and not specifically upon the activation of innate immune response (Citation19,Citation20), we believe that these cytokines may play a key role in the pathways underlying aortic wall rupture. To confirm further such a hypothesis, we did not find any significant differences between AAD and CAS in the values of other cytokines, such as VEGF, MCP-1, and TNF-α. These data are of particular interest since they seem to identify, in patients matched for the same traditional risk factors, a different pattern of inflammatory response, distinguishing breaking from athero-occlusive outcome. However, their involvement in several inflammatory conditions makes IL-6 and IL-8, but not MMP-12, useless as specific and distinguishing markers of Stanford-A AAD. In agreement with the literature we found that MCP-1 and VEGF levels did not differ between CAS and AAD, supporting the hypothesis that macrophage recruitment (Citation21,Citation22) and neoangiogenesis (Citation23,Citation24) are strongly related with any feature of the atherosclerotic disease. Intra-parietal activation of macrophages and vessel growth within the arterial wall, indeed, have been demonstrated to represent critical steps in AAD and plaque growth (Citation1,Citation2,Citation21–24).
Figure 1. Summary data of MMP-12 levels. Data are expressed as box plots representing the 25 and 75 percentiles, median, minimal, and maximal values. Statistical analysis: Mann–Whitney non-parametric test. *P < 0.05, **P < 0.01, ***P < 0.0001.
Finally, we would underline that, taken together, these results suggest that the different opposite outcomes of the atherosclerotic disease, in spite of a common primum movens and the sharing of some common inflammatory mechanisms, should follow different immunological pathways distinguishing the athero-occlusive outcome from rupture. It has been recently demonstrated that macrophages may be distinguished as two subpopulations, M1 and M2, which exert pro-inflammatory/matrix-degrading and regulatory/anti- inflammatory functions, respectively (Citation25); such differentiation has been demonstrated to be strongly influenced by a pro-inflammatory environment. It is well known that traditional cardiovascular risk factors act on the vessel wall directly as mechanical or chemical stressors (Citation26,Citation27), but also indirectly by promoting a pro-inflammatory state (Citation28). Thus, it is likely that in predisposed patients uncontrolled risk factors may induce such environmental modifications able to promote macrophage differentiation towards a lytic pattern, which leads to aortic rupture.
In conclusion, our results seem of particular interest since they identify MMP-12 as a specific marker of Stanford-A AAD in patients with no genetic predisposition. Furthermore, we confirm that, in AAD and CAS, macrophage activation represents a common denominator in the progression of the disease, although a distinguishing inflammatory/matrix- degrading pattern seems to influence the parietal response early on towards an occlusive or breaking outcome.
Declaration of interest: The authors have no conflict of interest to declare, including any specific financial, consultant, institutional, or other relationships. The authors have not received any honoraria from any companies or ‘for-profit’ organizations.
References
- Hansson GK. Inflammation, atherosclerosis and coronary artery disease. N Engl J Med. 2005;352:1685–95.
- Charo IF, Ransohoff RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med. 2006;354: 610–21.
- Del Porto F, Proietta M, Tritapepe L, Miraldi F, Koverech A, Cardelli P, et al. Inflammation and immune response in acute aortic dissection. Ann Med. 2010;42:622–9.
- Aukrust P, Otterdal K, Yndestad A, Sandberg WJ, Smith C, Ueland T, et al. The complex role of T-cell based immunity in atherosclerosis. Curr Atheroscler Rep. 2008;10:236–43.
- Weber C, Zernecke A, Libby P. The multifaceted contributions of leukocyte subset to atherosclerosis: lessons from mouse models. Nat Rev Immunol. 2008;8:802–15.
- Luo F, Zhou XL, Li JJ, Hui RT. Inflammatory response is associated with aortic dissection. Ageing Res Rev. 2009;8:31–5.
- Raman G, Moorthy D, Hadar N, Dahabreh IJ, O’Donnell TF, Thaler DE, et al. Management strategies for asymptomatic carotid stenosis: a systematic review and meta-analysis. Ann Intern Med. 2013;158:676–85.
- Touzé E. Treatment of carotid stenosis. Curr Vasc Pharmacol. 2012;10:734–8.
- World Health Organization. Definition and diagnosis of diabetes mellitus and intermediate hyperglycemia. Report of a WHO/ IDF Consultation. Geneva: World Health Organization; 2006.
- Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) Final Report. Circulation. 2002;106;3143–421.
- Mancia G, De Backer G, Dominiczak A, Cifkova R, Fagard R, Germano G, et al. 2007 ESH-ESC practice guidelines for the management of arterial hypertension: ESH-ESC Task Force on the Management of Arterial Hypertension. J Hypertens. 2007;25:1751–62.
- Curci JA, Liao S, Huffman MD, Shapiro SD, Thompson RW. Expression and localization of macrophage elastase (matrix metalloproteinase -12) in abdominal aortic aneurysms. J Clin Invest. 1998;102:1900–10.
- Longo GM, Buda SJ, Fiotta N, Xiong W, Griener T, Shapiro S, et al. MMP-12 has a role in abdominal aortic aneurysms in mice. Surgery. 2005;137:457–62.
- Ikonomidis JS, Jones JA, Barbour JR, Stroud RE, Clark LL, Kaplan BS, et al. Expression of matrix metalloproteinases and endogenous inhibitors within ascending aortic aneurysms of patients with Marfan syndrome. Circulation. 2006;114:I365–70.
- Newby AC. Metalloproteinase expression in monocyte and macrophages and its relationship to atherosclerotic plaque instability. Atheroscler Thromb Vasc Biol. 2008;28:2108–14.
- Koullias GJ, Korkolis DP, Ravichandran P, Psyrri A, Hatzaras I, Elefteriades JA. Increased tissue microarray matrix metalloproteinase expression favors proteolysis in thoracic aortic aneurysms and dissections. Eur J Cardiothorac Surg. 2004;26:1098–103.
- Patel MI, Melrose J, Ghosh P, Appleberg M. Increased synthesis of matrix metalloproteinases by aortic smooth cells is implicated in the etiopathogenesis of abdominal aortic aneurysms. J Vasc Surg. 1996;24:82–92.
- Lindeman JH, Abdul-Hussein H, Schaapherder AF, Van Bockel JH, Von der Thusen JH, Roelen DL, et al. Enhanced expression and activation of pro-inflammatory transcription factors distinguish aneurysmal from atherosclerotic aorta: IL-6 and IL-8 dominated inflammatory responses prevail in the human aneurysms. Clin Sci (Lond). 2008;114:687–97.
- Calcagni E, Elenkov I. Stress system activity, innate and T helper cytokines, and susceptibility to immune related diseases. Ann N Y Acad Sci. 2006;1069:62–76.
- Söderblom T, Oxhamre C, Torstensson E, Richter-Dahlfors A. Bacterial protein toxins and inflammation. Scand J Infect Dis. 2003;35: 628–31.
- Saraff K, Babamusta F, Cassis LA, Daugherty A. Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2003;23:1621–6.
- He R, Guo DC, Estrera AL, Safi HJ, Huynh TT, Yin Z, et al. Characterization of the inflammatory and apoptotic cells in the aortas of patients with ascending thoracic aortic aneurysms and dissections. J Thorac Cardiovasc Surg. 2006;131:671–8.
- Xu C, Zarins CK, Glagov S. Aneurysmal and occlusive atherosclerosis of the human abdominal aorta. J Vasc Surg. 2001;33:91–6.
- Holm PW, Slart RH, Zeebregts CJ, Hillebrands JL, Tio RA. Atherosclerotic plaque development and instability: a dual role for VEGF. Ann Med. 2009;41:257–64.
- Butcherand MJ, Galkina EV. Phenotypic and functional heterogeneity of macrophages and dendritic cell subsets in healthy and atherosclerosis prone aorta. Front Physiol. 2012;3:1–13.
- De Boer OJ, Van Der Wal AC, Teeling P, Becker AE. Leucocyte recruitment in rupture prone regions of lipid-rich plaques: a prominent role for neovascularization?Cardiovasc Res. 1999;41:443–9.
- Chae CU, Lee RT, Rifai N, Ridker PM. Blood pressure and inflammation in apparently healthy men. Hypertension. 2001;38:399–403.
- Schulz E, Gori T, Münzel T. Oxidative stress and endothelial dysfunction in hypertension. Hypertens Res. 2011;34:665–73.