Gut microbiota seems to be implicated in the atherosclerotic process. Translocation of bacterial endotoxins, like lipopolysaccharide (LPS) from gut to systemic circulation is being considered a new player contributing to chronic inflammation and atherosclerosis complications. Furthermore, endotoxins may have a role in platelet activation upon interaction with Toll-like receptor 4 (TLR4).
Until now, little is known about its role in the coronary thrombosis.
To explore this issue, we will compare LPS concentration and biomarkers of platelet activation in coronary thrombus and intra-coronary blood of patients with myocardial infarction and stable coronary disease respectively. Furthermore, to substantiate that LPS may be biologically active in the context, we will perform immune-histochemical analyses to investigate if TLR 4 is actually expressed at thrombus site
Serum levels of bacterial LPS, sP-selectin, a marker of platelet activation, and zonulin, a marker of gut permeability, will be measured in patients with ST-elevation myocardial infarction (STEMI) (n=50), stable coronary disease (n=50) and control subjects, matched for age, sex and cardiovascular risk factors (n=50). Serum LPS, soluble TLR4, a marker of TLR4 activation and sP-selectin will be also measured in coronary thrombi and coronary blood of patients with STEMI and stable coronary disease, respectively.
Finally, immuno-histochemical analysis will be performed to detect LPS and TLR4 activity in coronary thrombi.
If our hypothesis will be confirmed, LPS from gut microbiota could be be considered a novel and important trigger for platelet activation at the site of plaque rupture and a potential target for new therapies to counteract athero-thrombosis.
This study could suggest a role for bacterial endotoxins in contributing to thrombus growth.
Gut microbiota may represent e new and important element implicated in cardiovascular disease [1].
Gut microbiota, for instance, promotes the formation of a pro-atherogenic compound called trimethylamine-N-oxide, which is implicated in cholesterol accumulation in the macrophages and in forming foam cells [2]; clinical studies demonstrated that trimethylamine-N-oxide independently predicts cardiovascular disease and favor experimental thrombosis via platelet activation [3].
Lipopolysaccharide (LPS) is another component of gut microbiota, which may reach systemic circulation upon gut microbiota alteration and provoke systemic inflammation [4]. For example, LPS translocation into systemic circulation activates macrophages so favoring inflammation of visceral adipose tissue and hepatic Kupffer cells resulting in non-alcoholic fatty liver disease [5,6].
Other evidences in favor of the pro-atherogenic role of LPS include reduction of atherosclerotic plaque in animals with genetic deletion of Toll-like receptor 4 and acceleration of plaque formation in animals injected with lipopolysaccharide [7].
Less is known regarding the relationship between LPS and thrombosis. In vitro study showed that LPS could predispose to thrombosis by increasing thrombin generation via Tissue Factor up-regulation [8]. Recent studies focused also on the possibility that LPS could be a trigger for platelet activation. In vitro experiments using concentration of LPS in the same order of magnitude expected in coronary thrombi showed that LPS per se is unable to activate platelets but possess pro-aggregating property in presence of common platelet agonists; in particular, LPS were shown to dose-dependently stimulate platelet release of P-selectin and CD40L with a mechanism mediated by Nox2 activation [9].
In the present study, we will use two different assays to evaluate circulating levels of LPS in samples taken from coronary thrombi of STEMI patients and from intra-coronary blood of patients with stable angina to evaluate if a progressive increase of LPS exists from stable to unstable coronary disease. Moreover, we will evaluate the relationship between levels of LPS and platelet activation. Taking into account that platelet activation by LPS could be dose-mediated, we speculate that in STEMI patients, at the site of plaque rupture, LPS concentration would be enough to stimulate cells implicated in thrombus formation. To further address this issue, we will perform an immune-histochemical analysis to see if the receptor of LPS, namely Toll-like receptor 4, is up-regulated in coronary thrombi.
Implications of the study.
The presence of LPS in the coronary thrombi would suggest this glycolipid as another product from gut microbiota implicated in thrombus formation and would open potentially new avenues for anti-thrombotic treatment. New experiments could be planned to evaluate if the inhibition of Toll-like receptor 4 shows an anti-thrombotic effect in vivo. Moreover, serum LPS could become a new and valuable biomarker to stratify the risk of atherothrombosis and prospective studies could be planned to assess its prognostic value.
In conclusion, if our hypothesis will be confirmed, LPS from gut microbiota will be considered a novel and important trigger for platelet activation at the site of plaque rupture and a potential target for new therapies to counteract athero-thrombosis.
1. Cani PD, Osto M, Geurts L, Everard A. Gut Microbes 2012; 3: 279-88 10.4161/gmic.19625
2. Tang WH, Kitai T, Hazen SL. Circ Res 2017; 120: 1183-96 10.1161/CIRCRESAHA.117.309715
3. Zhu W, Gregory JC, Org E, et al. Cell 2016; 165: 111-24 10.1016/j.cell.2016.02.011
4. Piya MK, Harte AL, McTernan PG. Curr Opin Lipidol 2013; 24: 78-85 10.1097/MOL.0b013e32835b4431
5. Imajo K, Fujita K, Yoneda M, et al. Cell Metab 2012; 16: 44-54 10.1016/j.cmet.2012.05.012
6. Nobili V, Alisi A, Cutrera R, et al. Thorax 2015; 70: 769-81 10.1136/thoraxjnl-2015-206782
7. Lu Z, Zhang X, Li Y, Jin J, Huang Y. J Endocrinol 2013; 216: 61-71 10.1530/JOE-12-0338
8. Poitevin S, Cochery-Nouvellon E, Dupont A, Nguyen P. Thromb Haemost 2007; 97: 598-607
9. Cangemi R, Pignatelli P, Carnevale R, et al. J Infect 2016; 73: 107-14 10.1016/j.jinf.2016.05.013