Ischemic stroke (IS) occurs due to an embolus or thrombosis causing vascular occlusion in situ in certain brain parts (1). From the therapeutic point of view, the central zone of ischemia is irrelevant, since nerve cells cannot be saved. However, it is surrounded by penumbra, an area where the neurons are functionally disturbed, but structurally intact, with the possibility for the recovery of their function. On the other hand, without timely recanalization, an irreversible structural and functional damage in the zone of penumbra (ischemic necrosis) will occur. This has a significant effect on neurological damage, that is, on disability level (2). IS is the third leading cause of death and the leading cause of severe disability in the world (ref.) About 16 million people suffer of stroke in each year, with 5.6 million deaths. Around 85 – 90% of all stroke cases are people with IS. Risk factors of stroke include gender, age, race and ethnicity, as well as some different genetic factors, and can be modifying or non-modifying. Modifying group includes diabetes mellitus (DM), hypertension, atrial fibrillation, smoking, etc, also known as preventable risk factors (ref.) Dodati za našu zemlju.
The therapy of choice in the acute phase of IS is an intravenous administration of recombined tissue plasminogen activator (rt-PA). Rt-PA is the only licensed substance until now, that is recommended to be applied during the first 4 to 5h after IS in Europe, and within the first 3h in the United States. Studies have shown that an early application of this substance leads to a significant functional improvement and to a better recovery of patients with an IS. However, given that this therapy can cause potentially serious side effects, mostly hemorrhagic complications, the administration criteria are strictly defined (3). Tissue plasminogen activator (t-PA) is synthesized mainly in vascular endothelial cells and has a crucial role in fibrinolysis or blood clot dissolving. Alteplase is the first recombined form of t-PA and it is a serine protease like all the other rt-PAs. Alteplase has a catalytic activity, that is, to break the peptide bond between amino acids arginine and valine at position 560 and 561 inside the plasminogen. This way, plasminogen converts to plasmin, an active protolithic enzyme that hydrolyzes fibrin, fibrinogen and other proteins that form a blood clot. This leads to blood clot dissolution and vascular recanalization (4).
So far, a large number of genes which products are involved in processes of angiogenesis, coagulation/fibrinolysis and inflammation was identified (5). These genes have variations, such as Single Nucleotide Polymorphism (SNP), insertion/deletion variations (INDLs) or nucleotide polymorphisms. Gene polymorphisms are responsible for different levels of gene expression or gene products activity, and they have a direct influence on cell physiology and functionality, as well as on tendencies toward specific diseases, therapy response, pathogenesis, and in the end, on the disease outcome (6).
Renin-angiotensin system is a part of hormonal signaling, and angiotensin I-converting enzyme (ACE) is a significant enzyme in renin-angiotensin-aldosterone system and kinin-kallikrein system (ref.). ACE has a significant role in vascular remodeling, arteriosclerosis, but it’s also involved into pathogenesis of IS (ref.). ACE catalyzes the reaction that converts the inactive form of angiotensin (angiotensin I) into the active form (angiotensin II) which stimulates the regulation of vascular tonus (ref). ACE gene is located on the shorter arm of chromosome 17 (17q23) and consists of 26 exons. The insertion/deletion polymorphism is in the ACE intron 16. The insertion allele (I) has Alu sequence of 287bp, while the deletion allele doesn’t (ref.). The association between I/D polymorphism and coronary artery disease, myocardial infarction, and thrombosis was found (ref.) It has also been proven that the DD genotype is commonly associated with the cardiovascular events than the II genotype (ref). Apart from that fact, it is also linked to the appearance of leucoaraiosis, in cases of lacunar infarct (ref). According to some studies, in 56% of cases, subjects with DD genotype have an increased plasma ACE activity compared to subjects with II genotype (ref). The increased ACE level in plasma leads to decreased level of nitrogen oxide (NO), and lower vasodilatory response, and that, on the other hand, has a significant role in stroke incidence (ref).
The product of PAI-1 gene (plasminogen activator inhibitor 1) binds to t-PA, forming an inactive complex. In this way, PAI-1 acts as an inhibitor of endogenous fibrinolytic activity (11). Insertion-deletion polymorphism in the promoter region of PAI-1 gene (4G/5G) is associated with the plasma level of PAI-1. 4G allele variant is linked to the higher expression of PAI-1 gene, compared to 5G variant (12). On the other hand, the 4G/4G genotype is responsible for the poorer recovery of patients with IS treated with rt-PA (13).
Considering the fact that the importance of genetics has been already recognized in terms of treating and recovery of patients with stroke, and that the studies on this issue are scarce in our region, the aim of the following study was to…
Material and methods
Patients and data
This study involved 137 patients with IS and was treated with rt-PA therapy in Special Hospital of Cerebrovascular diseases “Sveti Sava”, Belgrade. Inclusion criteria were: patients with diagnosed IS who received rt-PA therapy and the age over of 18 years. Patients with cerebral infarction caused by subarachnoid hemorrhage and sinus venous thrombosis, as well as those who refused to give their written informed consent to participate, were excluded from the study.
. The potential neurological deficit in all patients was evaluated using The National Institutes of Health Stroke Scale (NIHSS) (ref), at hospital admission, 24 hours after rtPA therapy, at discharge and a month after IS. The severity of IS was determined according to NIHSS score at baseline as mild to moderate (score 5 – 10), moderate to severe (score 11 – 15) and severe damage (score over 15) (ref). Also, all patients were assessed using Modified Rankin Scale (mRS), at hospital discharge, 1 and 3 months after IS. We determined the favorable outcome as score 0–2 and poor outcome or death as score 3–6, according to literature (ref).
Data on social and demographic characteristics (age, gender, place of residence, education level), as well as on clinical characteristics (vascular risk factors – hypertension, diabetes mellitus, atrial fibrillation, heart failure, hyperlipidemia, smoking habit, ischemic heart disease; personal and family history of diseases; medication using) were obtained from medical records of patients, at baseline. Custom blood and biochemical tests were performed in all patients at admission. All patients received thrombolysis within 4 hours after onset of acute IS. The study was approved by the Ethical Committee of the institution and by the Ethical Commission of the Faculty of Medicine, University of Belgrade.
Patients were divided into two groups. The first group consisted of 80 consecutive patients who suffered from IS in the period of August 2016 to December 2017 and were admitted and treated in the facility. The second consisted of 54 patients who suffered from IS in the period of January 2015 to July 2016 and were retrospectively enrolled after they had written the informed consent. All these patients received rTPA therapy after the admission to the facility.
After 3 months all consecutive participants were contacted and mRS evaluation was performed. MRS evaluation was done by the specialist of neurology. MRSs in the second group were obtained using the medical records of patients at the moment of stroke occurrence and after discharge. Patients gave the information how they felt 3 months after stroke and those data were used to obtain mRSs at that particular moment.
Molecular genetic analysis
Total genomic DNA was extracted from peripheral blood leukocytes using a method known as salting out (ref). Fluorometry was used to determine the concentration and quality of the isolated DNA. The chain polymerization, also known as the PCR method, was used for the analysis of ACE-1 I/D polymorphism.
In this PCR reaction, primers were used to determine the insertion fragment. Primer sequence was Fw: 5′-CTG GAC ACC ACT CCC ATC CTT TCT -3′, and Rev primer sequence was: 5′-CTG GAC ACC ACT CCC ATC CTT TCT -3′.Conditions for the amplification were as follows: the initial denaturation at 95°C, in 35 3-step-cycles: a 1-minute denaturation at 94°C, hybridization at 63°C for 30 seconds, primer extension and DNA polymerization at 68°C for 1 minute and a final extension at 68°C for 7 minutes. Within the replicated part of ACE-1 gene that was 490bp long, there was an Alu sequence insertion 278 bp long, that corresponds to the I allele. The missing Alu sequence corresponds to D allele from 203bp, that is to say, it was 287bp shorter than the I allele. The products of the PCR replication were visualized and analyzed on 8% polyacrylamide gels colored with sybr®safe DNA gel stain color and illuminated in UV transilluminator.