Everolimus-Eluting Stents Reduce Monocyte Expression of Toll-Like Receptor 4

1 Cardiovascular Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 2 Cancer Research Center and Department of Pharmacology, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran. 3 Department of Pharmacology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 4 Medical Education Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 5 Hematology Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran.


Introduction
Atherosclerosis is a chronic and generalized disease of vessels. 1,2Smoking, hypertension, diabetes, and hypercholesterolemia are well-defined risk factors for Atherosclerosis. 3In line with other important components of atherosclerosis, mononcytes and T cells have distinguished role in initiation and progression of atherogenesis. 4 Macrophages can secrete cytokines, hydrolytic enzymes, metalloproteinases, and growth regulating molecules.][6][7] Toll-like receptor4 (TLR4) is a pattern recognition molecule, which consistent with its role in pathogen recognition, is expressed by cells involved in the first line of host defense, including neutrophils, macrophages, and dendritic cells.TLR4 sensitization on the surface of monocytes usually results in production of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α. 8- 10It is well established that TLR4 can be activated by a number of endogenous ligands that are formed in pathological conditions. 11It is also found that TLR4 is expressed in endothelial cells of human atherosclerotic lesions.It is suggested that TLR4 is involved in plaque formation and deterioration of the coronary arteries. 12,13t has been shown that patients with acute coronary syndromes (ACS) had an augmented expression of TLR4 on the surface of CD14 + monocytes compared to patients with stable angina. 14,15eperfusion therapies like coronary artery bypass graft surgery (CABG) or percutaneous coronary interventions (PCI) are used to treat patients with chronic stable angina who are still symptomatic although on routine medical therapy or have dangerous anatomical lesions. 15,16PCI contribution to local or general inflammatory responses is a subject of debate.Our previous works showed that implantation of stents could up-regulate the monocytic expression of TLR4. 17urrently most of PCI procedures are performed using DES.However, Bare Metallic Stents (BMS) are still in use.In general, anti-proliferative drugs are incorporated into DES to inhibit local cell proliferation and blood vessel occlusion.This study was designed to compare effects of DES and BMS on monocytic expression of TLR4 in patients who underwent PCI.Everolimuseluting stents are among the most popular stents used in PCI.Everolimus is an analogue of sirolimus and a macrolide antibiotic with immunosuppressive and antiproliferative activities.Everolimus inhibits cell proliferation and suppresses in-stent neointimal growth and significantly improve neointimal healing and reduce of neointimal proliferation. 18,19We focused on understanding whether in this setting everolimuseluted from the stents is able to reduce monocytic expression of TLR4.

Study patients
Between 2012 and 2014, patients with chronic stable angina undergoing elective percutaneous coronary intervention (PCI) in ShahidMadani Heart Hospital, Tabriz, Iran were included.Study was designed to include 95 patients who received DES and 95 patients who received BMS for chronic stable angina.Patient selection was done randomly.The exclusion criteria were previous myocardial infarction within 3 months, autoimmune diseases, complete chronic occlusion, advanced hepatic or renal disease, malignant neoplastic disease, receiving hydrocortisone during PCI, valvular heart disease, heart failure, unstable angina, receiving anti-inflammatory and immunosuppressive drugs and using antibiotics.The study was approved by ethical board of Tabriz University of Medical Science and informed consent letter was obtained from all participants.Cardiovascular risk factors, medications, sex, age and previous medical history were obtained by questionnaire.In addition to home medications, all of the patients were treated with standard PCI medications including heparin, aspirin and clopidogrel.

Blood collection and processing
Blood sampling was performed in a time dependent manner: time of admission (0 h), 2 hour and 4 hour (2h, 4h) after PCI procedure.Two ml blood was taken from patients and kept in EDTA anticoagulant tubes for flowcytometry.

Stenting procedure
Patients received intravenous heparin (10,000 U) prior the stenting procedure.Patients took aspirin (80 mg per day) and clopidogrel (75 mg per day) after implantation of stents.All PCI procedures were performed according to the routine protocols of ShahidMadani Heart Hospital, Tabriz, Iran.

Measurement of TLR4 expression on the surface of monocytes
Briefly, cells were stained for 40 minutes with monoclonal antibodies for human CD14 (BD, USA) conjugated with fluorescein isothiocyanate (FITC), and hTLR4 (BD, USA) conjugated with phycoerythrin (PE) at room temperature in the dark.FITC and PEconjugated non-specific mouse IgG2a antibodies were used for isotype controls (Dako, Denmark).Cellassociated fluorescence was measured using FACSCalibur flow cytometer (BD, USA).Data were analyzed using CellQuest software (BD, USA).

Statistical analysis
Data are presented as mean ± SD.Unpaired t-test and chi-square were used to compare differences between two groups.Statistical significance was considered as P<0.05.Analyses were performed using SPSS software version 16.

Characteristics of patients
Clinical and angiographic data of 190 patients are presented in Table 1.No significant difference was seen in the use of medications, white blood cell count or the risk factors.

Discussion
Everolimus is a proliferation signal inhibitor that acts on several cell types including vascular smooth muscle cells.The antiproliferative properties of the drug inhibit in-stent neointimal growth in experimental models and coronary vessels following stent implantation. 18,19verolimus is highly lipophilic, potent, and rapidly absorbed into tissue making it a desirable drug for intravascular delivery.
Evidence from literature suggests that inflammation has a critical role in re-stenosis following BMS or DES implantation. 20,21In this study, patients with chronic stable angina were included to compare effects of BMS and everolimus eluting stent as the DES on monocyte expression of TLR4 at different time intervals.We previously reported that PCI as an invasive method can positively regulate monocyte expression of TLR4. 17n contrast, some investigations demonstrated that various inflammatory cells were down-regulated following PCI procedure. 16In contrast, some investigations demonstrated that various inflammatory cells were down-regulated following PCI procedure.The study byTiong and colleagues revealed that neutrophil Mac-1 was deactivated and plasma matrix metalloproteinase-9 (MMP-9) was unchanged in patients that underwent PCI. 22enerally, at the time of stent implantation the overall inflammatory status is not equivalent in all patients.Our findings in link with other studies may partially explain the mechanism of restenosis after PCI in which inflammation plays a pivotal role.
In the present study, we found that both BMSs and DESs could significantly up-regulate CD14 + /TLR4 + monocytes after PCI procedure.However, BMS caused a greater upregulation of CD14 + /TLR4 + monocytes.In fact, DES contribution to partial suppression of local inflammation may be related to the relative down regulation of monocytic expression of TLR4 and of courses its related pro-inflammatory cytokines.
Reperfusion following a successful PCI can harm the myocardium through propagation of inflammation.Notably, Yang et al. proved that fibrinolytic therapy in patients with ST elevation MI could significantly upregulate monocyte expression of TLR4. 14vidence suggests that inflammatory responses following BMS are greater than DES.It is proposed that vascular injury by BMS can initiate a local inflammation. 23However, recent study showed that DES could cause a late increase in the levels of the proinflammatory cytokines compared with BMS. 24oreover, another study reported that sirolimus-eluting stents and paclitaxel -eluting stents are associated with higher risk of late thrombosis. 25This finding may partially explain the difference between BMS and DES in healing of the injured endothelial cells. 26he results of the present study provide evidence that eluted drugs in stents can partially suppress the early inflammation following PCI.The primary mechanism of action of such drugs is inhibition of T cell proliferation through blockade of the mammalian target of rapamycin (mTOR). 27However, the exact mechanism of action in this setting remains unknown.Further experimental and clinical studies should address concomitant use of medications that target PCI related inflammation.It is also beneficial that larger trials clarify a strong perception about the prognostic value of TLR4 in patients undergoing PCI.It is postulated that systemic use of immunosuppressive drugs can improve clinical outcome of patients following PCI or other craniological procedures that are associated with inflammation. 28,29nfortunately, systemic use of such drugs have had no considerable effect on clinical status of patients so far.Some limitation of our study should be acknowledged;Due to limited number of patients, our data should be confirmed in larger studies.Obviously our findings are representative for the majority of male patients with IHD in Iran.Finally, the activation mechanism of TLR4 in non-infectious conditions like PCI should be further investigated.

Table 1 .
Baseline and angiographic characteristics of patients