Downregulation of AnnexinA5 Gene Expression in Coronary In-Stent Restenosis – A Pilot Study

Background and aims: In- stent restenosis (ISR) is the Achilles heel of angioplasty. AnnexinA5 as an anticoagulant has been shown have anti-inammatory and anti-atherosclerotic effects. Here we aim to investigating the mRNA expression of AnnexinA5 in peripheral white blood cell of patients with in-stent restenosis. Methods Patients with the history of coronary stent implantation who candidate for re-angiography were entered the study and allocated into two groups according the results of re-angiography; in-stent restenosis (stenosis ≥ 50% in stent) and non-in-stent restenosis (stenosis < 50% in stent). Total RNA of WBC was extracted and cDNA was synthesized using commercial kits. AnnexinA5 expression was assessed with real time PCR and TaqMan probe and reported in relation with GAPDH as a housekeeping gene. AnnexinA5 expression was investigated in total 50 participants including 25 ISR and 25 non-ISR. Baseline characteristics including age, sex, smoking habits, hypertension, diabetes mellitus, dyslipidemia and stent in LAD were statistically the same in cases and controls. AnnexinA5 expression in ISR patients was 50% lower than controls.


Introduction
One of the most important methods which were developed for the treatment of coronary artery atherosclerosis is coronary angioplasty. It revolutionized the eld of cardiology. Coronary angioplasty reduced the need for coronary artery bypass graft (CABG) surgery for patients suffering from coronary artery disease (1,2). Angioplasty was rst performed in 1977 by Gruentzig (3).
Despite many advances in this eld and the rate of initial success and low complication rate, restenosis is considered as the Achilles heel of angioplasty and is a major clinical problem (4,5). In various studies, the rate of restenosis has been reported between 15 and 40% (6, 7). Different methods have been used to prevent restenosis. Stent placement is one of the most important ones that has reduced the rate of restenosis but in-stent restenosis (ISR) is one of the clinical problems in interventional cardiology with a success rate of 15 to 27%(8-10).
Various risk factors for ISR have been mentioned that can be divided into three groups: Patient-related Causes such as age, sex, genetics, and underlying diseases, Lesion -related Causes such as the length of the lesion, the location of the lesion and Procedure-related Causes such as number of stents, type of stent (11)(12)(13)(14).
Genetic backgrounds are one of the patients -related causes that are obtained by genome wide association studies (GWAS) or candidate gene study based on results of previous study or biological pathways related to restenosis (15,16). Evidence showed that factors which are inherited, can partly de ne reasons for the higher risk of restenosis incidence in patients after percutaneous coronary interventions. These agents include gene polymorphisms, which are the cause of various alterations in the gene products. Numerous studies have been focused on polymorphic alleles of genes encoding proteins related to restenosis (17). One of these proteins is AnnexinA5, a member of the Annexin family that was rst known as anticoagulant proteins (18). In addition to its inhibitory role in clot formation and thrombosis, its role as an anti-in ammatory and anti-atherosclerotic agent has been revealed in studies (19)(20)(21). Increasing amounts of atherosclerotic diseases and plaque vulnerability are the causes of vascular in ammation which can be adjust in order to prevent disease progression (22). AnnexinA5 which is abundant in atherosclerotic plaques binds reversibly, speci cally and with high a nity to externalized membrane PS (phosphatidyl-serine) of cells those initiating apoptosis and in ammation, result in PS-mediated platelet and leukocyte adhesion prevention, it has an impact on systemic in ammation and result in better endothelial function. In addition, anxA5 also exerts its anti-in ammatory role through its relevance to IFN-ϒ receptor, it can inhibit cellular in ammatory responses when IFN-ϒ release (23). Due to the important role of in ammation in restenosis, AnnexinA5 as an anti-in ammatory can play a role in restenosis.
Based on the results of a previous study by our research team, polymorphism of Anexina5 gene (ANXA5-1247T/C) was signi cantly related to coronary restenosis in recessive genetic model (24). In the present study, we investigated the expression rate of anxA5 in white blood cells of two groups including in-stent restenosis (ISR) patients in comparison with the none in-stent restenosis (NISR) cases.

Materials And Methods
Participants and sample collection: Total 50 patients who were referred for re-angiography because of ischemic coronary symptoms after previous angioplasty were enrolled to this study between December 2015 and June 2018. According to the results of the second angiography, 25 patients who had more than 50 percent stenosis in coronary stent were entered in the ISR (in-stent restenosis) group and 25 patients who had patent stent or lesser than 50 percent stenosis in coronary stent, were entered in the NISR (non in-stent restenosis) control group. The Scienti c Ethics Committee of the Medical University of Isfahan, Iran approved the study protocol (2015/930834) and all patients provided written informed consent.
Patients with chronic kidney disease (CKD), primary percutaneous coronary intervention (PCI), thrombophilia, active cancer, and autoimmune disease were excluded.
Extraction of RNA and synthesis of cDNA: 5 mL venous blood was collected in EDTA and RNase free tube from all cases for RNA extraction.
After buffy coat isolation from blood we normalized number of WBC by WBC counting using Sysmex XP-300 automated hematology analyzer and selected the sample contains approximately 10000 white blood cells for RNA extraction (The rst step of normalization) .Total RNA was extracted using TriPure Isolation Reagent kit (Roche, Germany) based on the manufacturer's instructions. The concentrations and purity of isolated RNA samples were checked by Nanodrop at 260 and 280 nm. cDNA Synthesis was performed using QuantiTect Reverse Transcription Kit (Qiagene, USA) according to the manufacturer's protocols.
We used GAPDH exon junction primer to con rm the cDNA synthesis, by this PCR reaction protocol: Initiation phase 94° C for 4 min, 35 cycles ampli cation phase 94° C for 60 s, 60° C for 60 s and 72° C for 45s , and nal extension phase at 72° C for 5 min. At last, the PCR products became visible on gel electrophoresis.

Relative quanti cation Real Time PCR:
The Anxa5 gene expression levels in ISR and NISR groups were measured by relative quanti cation real time PCR in Roche LightCycler96 instrument. We used predesigned Taqman Primer-Probe and TaqMan Universal Master Mixfrom Applied Bio system company (ABI, USA). The total reaction volume was 20 µl contained 10 µl TaqMan Universal Master Mix, 1µl (600ng/ µl) cDNA (the second step of normalization), 1µl (10 pmol/ µl) of AnxA5/GAPDH Taqman primer/probe and 8 µl ddH2O (Table1).
Real time PCR reaction according to ABI protocol was as following: The rst step preincubation: 50° C for 120s the second preincubation phase 95° C for 600s and 40 twostep ampli cation cycles: 95° C for 15 s, 60° C for 60 s. Because we used TaqMan probe there was no need to melting phase. GAPDH gene was used as internal control (the third step of normalization). All real time PCR reactions were done duplicated.

Statistical analysis:
The SPSS statistical software v22 (IBM, Chicago, IL) was used for statistical analyses. Continuous and categorical data were reported as mean ± standard deviation and percentage respectively. Kolmogorovsminrov test used to test the normality distribution of ΔCT .Mann-Whitney test was used to compare the expression of annexinA5 gene in the two groups of restenosis and non-restenosis, considering that the distribution of ΔCT in the two groups was not normal.

Results
Analysis for the expression of Anxa5 gene was performed in 25 in stent restenosis(ISR) cases and 25 non in stent restenosis(NISR) matched cases  To optimize the qPCR assay, we performed serial dilutions of a template and used the results to generate a standard curve. Based on the standard curve result the e cacy of PCR for both genes was acceptable and GAPDH gene was suitable for normalization (table3). The Comparative Ct method was used to compare AnnexinA5 gene expression between restenosis and non-restenosis group.
ΔCt of each sample was calculated using the formula Ct (AnnexinA5) -Ct (GAPDH). The normality of ΔCt distribution in case and control groups was measured using Kolmogorov-sminrov test, P value was <0.05 and distribution was out of the normal state, Mann-Whitney non-parametric test was used to compare the ΔCt of the two groups and P value was 0.034 means that expression of anxa5 gene in ISR and NISR group was signi cantly different.

Fold change calculating:
To compare the difference of AnnexinA5 gene expression between case and control groups we used the formula ΔΔ Ct =ΔCt ISR -ΔCt NISR.
Because of the approximately 100% e ciency of both primers (target and reference genes) in PCR, we used Livak method and 2 -ΔΔCt formula to calculate the fold change of AnnexinA5 gene expression in the ISR group in comparison to the NISR group. The fold change distribution was out of the normal state and the mean fold change was used to compare the change in gene expression, which was 0.05, meaning that the expression of the anxa5 gene in the ISR group was 50% lower than in the NISR group(Figure1).

Discussion
This study aimed to nd the expression of AnnexinA5 in white blood cells (WBC) of in-stent restenosis (ISR) patients compared to WBC of the non-in-stent restenosis (NISR) control cases.
As it was expected based on AnnexinA5 anti-in ammatory action, the main result of this study has shown a signi cant reduction (p =0.034) in the expression of AnnexinA5 in WBC of the in-stent restenosis patients compared to WBC of the non in-stent restenosis control cases. On the other hand, fold change calculations showed 50% reduction in the expression of AnnexinA5 in the ISR cases.
The role of AnnexinA5 as an anti-in ammatory agent has been shown in studies as there Ewing et al.
illustrated that its administration could diminish macrophage and leukocyte cohesion which led to the decrease in atherosclerotic development, macrophage presence and GRP78(endoplasmic reticulum stress marker) (21,25,26). Our study was the rst study on AnnexinA5 expression in WBCs and association to coronary restenosis. Ewing  and also observed increasing in T lymphocytes in atheroma tissue (27).
Because of the anti-in ammatory role of AnnexinA5, we observed down regulated of AnnexinA5 expression in patients with coronary restenosis, our study is consistent with the results of other studies.
Considering the high cost and complexity of coronary artery restenosis treatment (28), if physicians being able to predict the risk of patients coronary restenosis based on their genetic backgrounds (precision medicine) they will be able to select the appropriate stent type or reasonable treatment (angioplasty or open heart surgery) for patient.

Declarations
Funding: The authors would like to thank the Isfahan University of Medical Sciences, Isfahan, Iran for the nancial support (Grant Number: 1141292).

Con icts of interest/Competing interests:
We wish to con rm that there are no known con icts of interest associated with this publication and there has been no signi cant nancial support for this work that could have in uenced its outcome.
Availability of data and material: Not applicable Code availability: Not applicable Authors' contributions: BM: contributed in the conception of the work, conducting the study, revising the draft, approval of the nal version of the manuscript.
SSS: contributed in the conception of the work, conducting the study, data analysis for the work.
MS: contributed in the conception of the work, conducting the study, revising the draft.
MM: contributed in the conception of the work, conducting the study, data analysis for the work.
IO: contributed in the conception of the work, conducting the study, revising the draft.
RM: contributed in the conception of the work, conducting the study, revising the draft.
PA: contributed in the conception of the work, conducting the study, data analysis for the work.
GMM: contributed in the conception of the work, conducting the study, revising the draft.
SMH: contributed in the conception of the work, conducting the study, revising the draft, approval of the nal version of the manuscript, and agreed for all aspects of the work.
Ethics approval: The study protocol (2015/930834) was approved by the Ethics Committee of the Medical University of Mashhad, Iran.

Consent to participate:
The Scienti c Ethics Committee of the Medical University of Isfahan, Iran approved the study protocol (2015/930834) and all patients provided written informed consent.
Consent for publication: All authors are satis ed with the publication of the article in the Molecular Genetics and Genomics journal