Dissertation committee:
Χρήστος Τσιγκρής, Ομότιμος Καθηγητής, Ιατρική, ΕΚΠΑ
Κωνσταντίνος-Θεόδωρος Λιακάκος, Καθηγητής, Ιατρική, ΕΚΠΑ
Σωτήριος Γεωργόπουλος, Καθηγητής, Ιατρική, ΕΚΠΑ
Χρήστος Κλωνάρης, Καθηγητής, Ιατρική, ΕΚΠΑ
Κωνσταντίνος Φίλης, Καθηγητής, Ιατρική, ΕΚΠΑ
Ιωάννης Καραβοκυρός, Αναπληρωτής Καθηγητής, Ιατρική, ΕΚΠΑ
Χρήστος Μπακογιάννης, Αναπληρωτής Καθηγητής, Ιατρική, ΕΚΠΑ
Summary:
Introduction: Since its discovery in 1926, relaxin has been regarded as a peptide hormone that plays a role in the enlargement of pubic symphysis during the perinatal period. Since then, it has been found to exhibit a variety of biological functions in various organs and systems, especially the cardiovascular system. The specific mechanisms through which it exerts its effects on the cardiovascular system have not yet been elucidated. What is known is that of the three types of relaxin found in humans (RL1, RL2, RL3), the major isoform is relaxin 2, commonly referred to as "relaxin". Relaxin family peptides target specific G protein-coupled receptors (G-coupled receptors), defined as relaxin family peptide receptors (RXFP), of which RXFP1 is the specific receptor for RL2. RL2 has been found to exert some of its biological activity through intracellular nitric oxide (NO), which is a potent vasodilator and its lack or production is a cause of atheromatosis according to modern atherogenesis theories. In other studies it has been found to affect the synthesis of metalloproteinases (MMPs) 2 and 9 in various tissues including the arteries. The role of metalloproteinases is well known in modern theories for aneurysm replacement because of their action on the destruction of collagen fibers and elastin in the aneurysm wall. Therefore, there is a theoretical background linking relaxin to vascular disease, but to date it has only been studied in experimental animals and not in humans.
Aim: The purpose of the present study was to investigate the amount of relaxin released in the serum and the expression of the relaxin gene in arterial preparations of patients with atherosclerosis and arterial aneurysm, in relation to the type and severity of vascular disease, as well as in control subjects without evidence of vascular disease. Meanwhile, the potential mechanisms of action of relaxin will be elucidated by simultaneously measuring the expression of MMP-2, MMP-9 and eNOS in arterial preparations. Our aim is to demonstrate any predictive value of relaxin in the presence and severity of vascular disease, as well as the possibility of using relaxin as a biological indicator of early diagnosis of aneurysm and its potential therapeutic applications in atherosclerosis.
Experimental design: Comparative observational study
Location: 1st Clinic of Surgery, National and Kapodistrian University of Athens - Center for Experimental Surgery, Institute of Biomedical Research, Academy of Athens.
Subjects: Men and women regardless of age and race undergoing open surgical repair of arterial aneurysm, extracranial carotid disease or peripheral artery disease, classified according to the clinical severity of vascular disease. Healthy blood donors, patients undergoing temporal artery biopsy and cadaveric organ donors / accident victims with no history or clinical evidence of arterial aneurysm, peripheral artery disease or extracranial carotid artery disease were used as controls.
Method: The 37 patients treated for vascular disease were categorized into two groups according to the nature of their arterial pathology and each group was further categorized into three subgroups according to the clinical severity of arterial disease, as follows: Atherosclerosis group (ATH group) (n = 21) who underwent surgery for atherosclerotic disease and were further categorized according to the clinical severity of their arterial disease in three subcategories: 1) Atherosclerosis Group 1 (AT1, n = 10): Patients with clinical implications of moderate severity, ie asymptomatic stenosis> 70% of internal carotid artery or severe Rutherford stage 3 intermittent claudication, 2) Atherosclerosis group 2 (AT2, n = 6): Patients with severe functional clinical consequences, ie transient ischemic stroke and stenosis> 50% of internal carotid or Rutherford stage 4 lower extremity ischemic pain and 3) Atherosclerosis Group 3 (n = 5) Patients with permanent clinical disability due to atherosclerosis, ie ischemic stroke and stenosis of> 50% of the internal carotid artery or major / minor ischemic tissue loss of the lower end stage 5 and 6 Rutherford. Arterial aneurysm group (n = 16) who underwent surgical aneurysm repair and were further categorized based on the diameter of the aneurysm and their clinical picture upon admission to the hospital in three subcategories: 1) Arterial aneurysm group 1 (AA1, n = 5): Patients with asymptomatic aneurysm 250-300% of normal artery diameter, 2) Arterial aneurysm group 2 (AA2, n = 4): Patients with asymptomatic aneurysm 300% of normal arterial diameter 3) Arterial aneurysm group 3 (AA3, n = 7): Patients with symptomatic aneurysm or asymptomatic aneurysm diameter> 350% of normal artery diameter. The design of the study provided for the creation of three control groups. In the first control group, patients were selected for probation of prolonged fever and underwent temporal artery biopsy (TAB group, n=6). The second control group of all vascular diseases were healthy blood donors (HBD group, n=10). The third and final control group included cadaveric organ donors, as well as victims of accidents (Autopsy group - AG group, n=10) regardless of gender, age or race, who either underwent part of their abdominal aorta during organ donation (cadaveric donors) or during autopsy (accident victims). Both ATA, AA and TAB groups received both blood and arterial tissue samples. Blood samples were obtained from the HBD group, and arterial tissue samples were obtained from the AG group. ELISA was used to determine the amount of RL2 circulating blood serum, while qRT-PCR was used to determine the amount of RL2, MMP-2, MMP-9 and eNOS mRNA.
Results: The serum RL2 of patients with arterial aneurysm, with an indication for treatment, is significantly increased compared to patients and controls who have no history or clinical evidence of aneurysm. The fluctuation of serum RL2 is proportional to the size of the aneurysm and the presence of symptoms therefrom and is significantly increased in patients with aneurysm> 350% of normal diameter or in symptomatic aneurysms compared to smaller or asymptomatic aneurysms. The gene expression of RL2 and MMP-2, MMP-9 and eNOS in aneurysmal arteries> 350% of normal diameter or in symptomatic aneurysms is significantly increased than that observed in smaller aneurysms, atherosclerotic plaques, or normal arteries. Expression of relaxin is not correlated with metalloproteinases in larger and symptomatic aneurysms, suggesting an independent role for RL2 in arterial wall integrity and remodeling in aneurysm. In addition, a negative association of RL2 with MMPs was observed in smaller aneurysms, but not in larger aneurysms, suggesting the involvement of other mechanisms in this multifactorial disease. On the other hand, circulating serum RL2 was increased in the stages of atherosclerosis with mild clinical impact (subclasses AT1 and AT2) and gradually decreased as the disease worsened clinically. At the same time, RL2 increases its expression in atherosclerotic plaques and serum levels, acting as a physiological compensatory mechanism for the decreased blood supply to the milder clinical stages of atherosclerosis, but this mechanism is ameliorated in advanced clinical stages of the disease.
Conclusions: RL2 is a peptide that is eligible to be used as a biomarker of aneurysm disease. To confirm the clinical utility of RL2, studies involving patients with smaller aneurysms are also required to determine the reference values above which the general population is likely to develop arterial aneurysms and lead to further selective imaging investigation and intervention based on existing evidence. Finally, the fact that serum RL2 is significantly elevated in symptomatic aneurysms indicates that it may be useful in monitoring patients with smaller aneurysms and in determining which of these patients are at risk of rupture. The fact that RL2 has a negative association with MMP-2 in mild atherosclerosis (subcategory AT1) and a positive association with eNOS in moderate arteriosclerosis (subcategory AT2) indicates that RL2 has a positive effect and could potentially be used therapeutically in atherosclerosis. RL2 may be related to coronary heart disease and its clinical severity, but the small number of patients does not allow any firm conclusions. A study of the serum levels of RL2 in patients with coronary heart disease, classified into subgroups according to the clinical severity of the disease (for example, stable angina, unstable angina, and myocardial infarction) according to our study standards, could answer this question.
