Biomarkers & Inflammatory markers of atherosclerosis

As atherosclerosis is a complex inflammatory disease, there are many influential biomarkers that contribute to the disease’s progression as well as biomarkers from atherosclerosis related diseases such as periodontal disease and autoimmune diseases i.e. Diabetes, RA, SLE. Biomarkers can be proteins, DNA and mRNA which are measured to asses biological, pathological processes and pharmacological responses. Biomarkers can be classified as early, predictive and prognostic biomarkers depending on disease stage (Huang et al., 2010).

Identifying biomarkers for disease and finding therapeutic targets is crucial to research and treatment and overall a populations mortality and morbidity rate (Uno and Nicholls, 2010). A good approach in investigating biomarkers in a progressive disease such as atherosclerosis that may take decades to show symptoms would be to identify separate markers associated with the different stages of the disease. However ongoing research has particularly focused on some of the following pro and anti-inflammatory mediators. Atherosclerotic associated cells as discussed earlier: ECs, SMCs, and macrophages are all producers of important biomarkers.

IL-10 is classified as a pleiotrophic anti-inflammatory lymphokine that suppresses expression of IFN-gamma, TNF- alpha, GM-CSF and proliferation of T cells. For the immune system to be fully functional there must be a balance of pro and anti-inflammatory molecules in systemic circulation. IL-10 deficiency has been reported in numerous disease associated with inflammation including RA and SLE (Pyo et al., 2003) and UAS, patients demonstrate an imbalance of TNF-α and IL-10 levels in serum (Waehre et al., 2002). IL-10 deficiency in atherosclerosis leads to an increase in fatty streak formation and proteolytic and coagulant activity (Caligiuri et al., 2003) as it inhibits Th1 cytokine production (Fiorentino et al., 1991), reduces CC chemokine production that would result in leukocyte homing and down regulates ICAM-1. It also contributes to prevention of plaque destabilization by reducing MMP production. However, like other cytokines involved in atherosclerosis, serum levels of IL-10 in patients vary between studies and disease type (Stenvinkel et al., 2005). IL-10 plays a cross-regulatory role with IL-12, and inhibits its oxLDL induced release (Uyemura et al., 1996).

TNF-alpha is a pleiotrophic pro-inflammatory cytokine and it is mainly exported by monocytes and macrophages. TNF signals through the TNF receptor (TNFR) (Fragoso et al., 2013), TNFR is found on endothelial cells and ultimately activates NF-Kappa Beta which is responsible for cell survival, proliferation, inflammation and immune regulation. Many inflammatory marker genes consist of an NF-kappa beta promoter region, therefore if protein expression is regulated by TNF-α it may be considered as a pro-inflammatory marker. It is involved of all stages of atheroma formation: initiation, development, susceptibility, severity and response to treatment (Fragoso et al., 2013). TNF-α is involved in the production of chemokines (IL-6, CRP) and cytokines, expression of adhesion molecules (ICAM-1, VCAM-1), recruitment of leukocytes, induction of smooth muscle cell proliferation and lipid metabolism (decreases HDL activity) (Bruunsgaard et al., 2000). TNF-α concentration is 200 times higher in intimal thickenings of atherosclerotic aortas than in corresponding serum samples (Rus and Vlaicu, 1991). A study carried out on patients suffering from RA and AS treated with anti-TNF-α for 1 year had a significant reduction in aortic stiffness (Angel et al., 2012).

IFN-γ is the only type II interferon, type I interferons include α, β and δ. Cells which produce IFN-gamma include activated T lymphocytes (CD4+ Th1 cells), natural killer cells, monocytes/macrophages , dendritic cells and B cells. CytokinesIL-12 and IL-18 released by antigen presenting cells activate IFN-γ secretion from these cells. IFN-gamma has multiple pro-atherogenic roles. IFN-gamma signalling activates immune cells such as T-cells, macrophages and NK cells, Class I and II major histocompatibility complex (MHC) molecules, cytokine production and increased expression of adhesion molecules and chemokines at site of lesion. (Harvey and Ramji, 2005, Tenger et al., 2005). IFN-gamma is known as a pro-inflammatory cytokine but is likely to play both pro and anti-inflammatory role in atherosclerosis (Muhl and Pfeilschifter, 2003). IFN-gamma is involved in multiple stages of atherosclerosis as a pro-inflammatory cytokine: foam cell formation, immune response and plaque development (McLaren et al., 2009). Therapeutics focusing only on this cytokine have vast potential in prevention of atheroma development, a study demonstrating IFN-gamma neutralisation by a plasmid encoding soluble IFN-γR reduced lesion advances in mouse models (Gotsman and Lictman, 2007).

IL-6 is a glycoprotein produced by monocytes/macrophages, adipose tissue and endothelial cells. IL-6 stimulates SMC proliferation, MCP-1 secretion from macrophages and ICAM-1 expression on ECs. Elevated IL-6 plasma concentrations have been associated with morbidity and mortality rates in unstable angina (Koenig and Khuseyinova, 2007). In carotid artery and coronary artery patients, IL-6 had significantly higher expression levels than controls and correlated with the lumen diameter of the common carotid and was considered a useful biomarker (Larsson et al., 2005). IL-6 and IL-8 have shown significantly higher levels in fibrous plaque than the normal arterial wall (Rus et al., 1996).

IL-2 is a known angiogenic factor which has multiple inducers, including IL-4, IL-6, TNF-alpha and itself. It has been shown to be increased in coronary artery disease (CAD) and stable angina (SA) patients but not acute coronary syndrome (ACS) (Ozeren et al., 2003). In a study on carotid endarterectomise by Frostegard et al., the imbalance of pro and anti-inflammatory cytokines were significantly different, IL-2 and IFN-gamma were present in 30-50% of plaques (Frostegard et al., 1999).

GM-CSF is a cytokine responsible for the differentiation progenitor cells into mature granulocytes and macrophages and the proliferation of DCs (Alberts-Grill et al., 2013). It is a key mediator in the response to injury phase of inflammation initiation. Protein and mRNA studies have confirmed the presence of GM-CSF in non-diseased arteries and its up-regulation in atherosclerotic human coronary arteries (Plenz et al., 1997). The expression of GM-CSF in diseased arteries can be due to the excessive amount of oxLDL, which induces cytokine release by endothelial cells. The ability of GM-CSF to regulate granulocytes and macrophages contributes to plaque progression (Shaposhnik et al., 2007).

Of the IL-1 family IL-1 beta is the most predominant isoform circulating in humans. Vascular cells can both produce and be targets of IL-1β signalling. IL-1β signals through the MAPK pathway by binding to IL-1RI (receptor) and activates TFs and NF-ĸβ which results in pro-inflammatory gene expression (Chamberlain et al., 2006). In atherosclerosis, IL-1β is considered a significant contributor in all stages of the disease; it increases adhesion molecule expression, vascular permeability and SMC proliferation (Apostolakis et al., 2008).

IL-12 is an important regulator of Th1 and Th2 cell responses. It is produced by activated monocytes and is a T cell growth factor. IL-12 expression may be initiated by monocyte activation by oxLDL but not minimally modified (MM)-LDL. IL-12 p40 mRNA and IL-12 p70 protein have been found in abundance in atheroma (Uyemura et al., 1996).

Il-18 is a member of the IL-1 family alongside IL-1β. A wide range of cells express this pro-inflammatory IFN-γ inducing cytokine such as monocytes/macrophages, T and B cells, dendritic cells and epithelial cells (Apostolakis et al., 2008). IL-18 gene is found on chromosome 11: position 11q22.2-q22.3 (Okamura et al., 1995).

IL-18 is associated with inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus and skin diseases, psoriasis and atopic dermatitis (Sims and Smith, 2010). Unlike most cytokines, IL-18 is constitutively expressed without a stimulant. In atherosclerosis, IL-18 has many roles; it induces cytokines GM-CSF, TNF-α, IL-1β, IL-6 and chemokine IL-8, which significantly contribute to chronic inflammation at the site of lesion development. The most studied influence IL-18 has on immune cells is its ability to stimulate a potent amount IFN-γ from Th1 cells. A combination of IL-12 and IL-18 also generates IFN-γ on a wider range of cells, CD8+ T cells, B cells and NK cells (Arend et al., 2008).

IL-18 studies have shown the presence of IL-18 co-localized with macrophages in atherosclerotic lesions while absent in healthy arterial regions. In mouse models, IL-18 enhances atherosclerosis through IFN-γ release (Whitman et al., 2002). Also, elevated levels of IL-18 in carotid and aorta atheromas correlated with the increasing potential of plaque destabilization. However studies show conflicting results describing the role of IL-18 as pro- or anti-atherosclerotic, depending on the experimental model (Arend et al., 2008). IL-18 has been to shown to impact on elevating CXCL16 expression in lesions along with IFN-γ (Tenger et al., 2004). In aortic smooth muscle cells CXCL16 dependent ASMC proliferation is mediated by IL-18 (Chandrasekar, et al., 2005). Apart from immune cells IL-18 also has an effect on endothelial cell expression, IL-18 upregulates adhesion molecules ICAM-1 and VCAM-1 (Apostolakis et al., 2008).