Introduction

Relaxin 2 (RL2) is a peptide hormone that acts on various organs and systems, particularly the cardiovascular system1. RL2 is structurally related to insulin, and like insulin, its mature peptide derives from a pro-hormone after processing by prohormone convertases2,3,4. RL2 binds to and activates relaxin family peptide receptor-1 (RFXP-1), a G protein-coupled receptor (GPCR) that triggers signaling pathways through the generation of second messengers such as nitric oxide (NO) and cyclic adenosine monophosphate (cAMP) and the stimulation of the phosphorylation of mitogen-activated protein (MAP) kinases3,4,5,6. Interestingly, reduced NO synthesis and bioavailability due to direct quenching by superoxide anion can contribute to cardiovascular diseases such as atherosclerosis7.

Apart from reproductive tissues, RL2 has been detected in human brain and heart3,4, and RFXP-1 is expressed in rodents in the aorta, vena cava, mesenteric artery, mesenteric vein, femoral artery, femoral vein, small pulmonary arteries and small renal arteries3,8,9. Meanwhile, RL2 upregulates the synthesis of matrix metalloproteinase (MMP)-2 and -9 in various tissues, including arteries10,11,12. The role of MMPs in aneurysm formation is well established13,14,15,16,17. MMPs induce the degradation of the vessel wall extracellular matrix. In that context, RL2 may theoretically be implicated in various vascular diseases; yet, current literature on its role in atherosclerosis and aneurysm formation is scarce18,19,20,21,22.

The aim of the present study was to investigate the level of serum RL2 in patients with an arterial aneurysm (AA) and correlate them with the size of the aneurysm and the related clinical presentation of the patient (whether symptomatic or not). We also investigated whether serum RL2 in atherosclerotic (ATH) patients was correlated with the clinical severity of the disease (permanent or not ischemic manifestations from organs that are supplied from the atherosclerotic vessels). Both AA and ATH patients were compared with control groups consisting of healthy blood donors (HBD) and patients undergoing temporal artery biopsy (TAB).

Materials and methods

Study population

A total of 53 subjects were enrolled in our study; 37 patients were scheduled to undergo surgery at Laiko Hospital in Athens, Greece, whereas 6 patients undergoing temporal artery biopsy (TAB) and 10 healthy blood donors (HBD) served as the control groups. The study was conducted in accordance to the principles outlined in the Declaration of Helsinki, and all subjects signed an informed consent for the use of their clinical and laboratory results for scientific purposes under the condition of anonymity. In brief, our interventional group consisted of 21 patients undergoing surgery for different forms of atherosclerotic disease and 16 patients undergoing surgery for an arterial aneurysm.

Study groups and sub-categorization

ATH group

The ATH group included patients with extracranial carotid artery disease (n=12) or peripheral arterial disease (n=9). These patients were further subcategorized according to the clinical severity of their disease into three groups: 1) atherosclerosis group 1 (ATH1, n=10): patients with moderate functional consequences [asymptomatic internal carotid stenosis >70% or claudication (Rutherford stage 3)]; 2) atherosclerosis group 2 (ATH2, n=6): patients with severe functional consequences [transient ischemic attack or rest pain (Rutherford stage 4)]; and 3) atherosclerosis group 3 (ATH3, n=5): patients with permanent disability [stroke or tissue loss (Rutherford stage 5–6)]. In patients with extracranial carotid artery disease, an experienced neurologist determined neurologic status, whereas the degree of carotid stenosis was determined by the appropriate imaging modality [digital subtraction angiography (DSA); computed tomographic angiography (CTA); or magnetic resonance angiography (MRA)] using the NASCET method23. Patients with peripheral arterial disease were categorized according to the revised version of recommended standards for reports dealing with lower-extremity ischemia24.

AA group

AA group patients had an abdominal aortic aneurysm (AAA) (n=13), thoracic aortic aneurysm (TAA) (n=1), internal iliac aneurysm (IIA) (n=1) or popliteal aneurysm (PA) (n=1) and were further categorized according to the size and clinical presentation of their aneurysm into three subgroups: 1) aneurysm group 1 (AA1) (n=5): patients with an asymptomatic aneurysm of a diameter 250%–300% of that of the normal artery; 2) aneurysm group 2 (AA2) (n=4): patients with an asymptomatic aneurysm of a diameter 300%–350% of that of normal artery and 3) aneurysm group 3 (AA3) (n=7): patients with either a symptomatic aneurysm of any diameter or an asymptomatic aneurysm of a diameter >350% of that of normal artery. Representative diameters of normal adult arteries as they are presented in the suggested standards for reporting on arterial aneurysms25 were used for AA group categorization. Aneurysm size was determined by CTA or MRA, and a patient was categorized as symptomatic based on his/her symptoms alone, regardless of the existence of rupture. Although we are not aware of any report using the above-mentioned categorization, this approach was utilized since aneurysm size is by definition proportional to the diameter of normal artery. In that context, all patients in the AA group had dilatation of various arterial segments, while the clinical severity of the disease was directly related to the presence of symptoms.

TAB and HBD control groups

TAB group patients (n=6) were admitted for prolonged fever, and after an extensive diagnostic work-up, a temporal artery biopsy was deemed necessary. Exclusion criteria were a pathology report of temporal arteritis and a history or clinical signs of arterial aneurysm, extracranial carotid artery disease or peripheral arterial disease. Finally, the HBD group (n=10) included healthy individuals participating in blood donation on a voluntary basis without history or clinical signs of an arterial aneurysm, extracranial carotid artery disease or peripheral arterial disease.

Blood analysis

Blood samples were collected by venipuncture after subjects were fasted overnight. In patients undergoing emergent operation, such as ruptured aneurysm repair and life-saving amputation, the blood samples were collected during induction of anesthesia. The samples clotted for 30 min at room temperature before centrifugation for 15 min at 1000×g at 4 °C (Hettich Rotina 35R centrifuge; Hettich GmbH & Co KG, Tuttlingen, Germany). Finally, serum was collected and stored at −20 °C until analysis was performed in duplicate; dilution was assessed as per protocol. Using a commercially available enzyme immunoassay kit (DRL200, R&D systems, Minneapolis, USA), we measured serum RL2 according to the protocol of the manufacturer with an ELISA reader system (Spectramax 190; Molecular Devices, Sunnyvale, Calif, USA). Full blood count and clinical chemistry analysis were determined using a standard method (Sysmex XE-5000, Sysmex Europe GmbH, Norderstedt, Germany and Siemens Advia 1800, Siemens Healthcare GmbH, Erlangen, Germany).

Statistical analysis

The results are presented as the mean±standard deviation (SD). Statistical significance for two-group comparisons was calculated using Student's unpaired-samples t-test. One-way ANOVA was performed for multiple comparisons between measured parameters. A P-value <0.05 was considered statistically significant. All statistical calculations were performed using GraphPad Prism version 4.03 (GraphPad Inc, CA, USA).

Results

Baseline characteristics

Demographics, smoking status, current medication and concurrent diseases of the study cohort are summarized in Table 1. Apart from the HBD group (mean age: 43.5±3.41), all subjects underwent baseline preoperative hematologic and biochemistry tests, including full blood count, renal and liver function tests. Renal and liver tests were normal in all groups, and none of the patients presented with acute heart failure. All patients were stable without signs of septic or hypovolemic shock, even in emergent cases of gangrene or aneurysm rupture, with only two exceptions. One female patient (ATH group) undergoing emergent above-knee amputation was septic during blood sample collection. Additionally, a male participant from the AA group, with a history of kidney transplantation, had mild renal function impairment and presented with a ruptured aneurysm of his left common iliac artery. In both cases, no differences were found in serum RL2 compared to the other patients in their respective subgroups.

Table 1 Subject baseline characteristics.
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Overall, AA, ATH and TAB groups were matched for gender, age, medication, history as well as renal and liver function tests. The HBD group, although matched with the ATH and AA groups for gender and smoking status, represented a different population group that was approximately 25 years younger. As defined by the inclusion criteria, no one in the HBD group had a history of concomitant disease, and no one received any medication.

Serum RL2 in all study groups

The AA group's serum RL2 (49.39±8.620 pg/mL) was significantly increased compared to the ATH group (16.22±4.697 pg/mL, P<0.01), TAB group (15.86±4.286 pg/mL, P<0.01) and HBD group (10.32±1.354 pg/mL, P<0.01) (Figure 1). No significant difference was observed between ATH and TAB (P>0.05). Both ATH (P<0.01) and TAB (P<0.01) patients had significantly increased serum RL2 compared to HBD.

Figure 1
figure 1

Relaxin 2 serum levels in all study groups. Group definitions: TAB (temporal artery biopsy); ATH (atherosclerosis); AA (arterial aneurysm); HBD (healthy blood donors). *P<0.05 between groups; #P<0.05 between groups (TAB, ATH, AA) and HBD. Data are presented as the mean±SD.

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Serum RL2 in subcategorized study groups

Further statistical analysis in AA patients after categorization into subgroups, as shown in Figure 2, revealed that serum RL2 increased proportionally to the size and the severity of the clinical presentation of the patients. The AA1 group (38.06±2.7 pg/mL) had considerably lower serum RL2 compared to the AA2 group (49.99±2.1 pg/mL, P<0.01); and AA3 group (57.1±1.1 pg/mL, P<0.01). All AA subgroups had significantly higher serum RL2 than all ATH subgroups (P<0.01), TAB patients (P<0.001), and HBD group (P<0.01).

Figure 2
figure 2

Relaxin 2 serum levels in subgroups. Serum relaxin 2 increased with the size or symptom status of the aneurysm, while it decreased with the clinical progression of atherosclerosis. Data are presented as the mean±SD. *P<0.05 between groups (ATHx, AAx) and TAB; #P<0.05 between groups (ATHx, AAx) and HBD.

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Further statistical analysis in the ATH subgroup showed that serum RL2 level was inversely correlated with the clinical severity of atherosclerosis; RL2 was higher in the ATH1 (20.65±1.419 pg/mL) compared to the ATH2 group (13.61±2.027 pg/mL, P<0.01) and the ATH3 group (10.50±0.6044 pg/mL, P<0.01). In patients with permanent ischemic damage from target organs (ATH3 group), serum RL2 was comparable to HBD (P=0.788). The TAB group's serum RL2 was comparable to the ATH2 group (P>0.05).

Discussion

During the last two decades, several biomarkers have been investigated for a potential relationship with the development and progression of aneurysms in an effort to optimize timely diagnosis26,27. Matrix metalloproteinases, MMP-9 in particular, are among the most extensively studied biomarkers28. The relationship between MMPs and RL23,4,10 was the rationale behind the present study in patients with an arterial aneurysm. It was evident that patients with a known AA requiring surgical treatment had substantially higher serum RL2 than ATH patients or control subjects. In addition, serum RL2 increased proportionally to the increase in aneurysm size, exhibiting the highest levels in either a symptomatic aneurysm of any diameter or an asymptomatic aneurysm of a diameter >350% of that of the normal artery. To the best of our knowledge, this is the first study to correlate the serum RL2 level with the size and existence of an aneurysm.

There have been a few reports suggesting the involvement of RL2 in the atherosclerotic process13,18,20,21,22,23, showing a potential beneficial effect on vascular inflammation21, vascular oxidative stress, vasorelaxation, and atherosclerotic plaque development23. Based on the current knowledge, we hypothesized that RL2 would be decreased in atherosclerotic patients to a level significantly lower than that of healthy individuals. This hypothesis was not verified; on the contrary, we found that serum RL2 was higher at early stages of atherosclerosis and gradually decreased with the disease progress, eventually exhibiting comparable results between patients with permanent ischemic manifestations from target organs (ATH3 group) and HBD. Interestingly, a previous study showed beneficial results from oral administration of porcine relaxin in patients with peripheral artery disease14. Based on that, our findings could imply that RL2 compensates for the decreased blood supply in the early stages of atherosclerosis, a mechanism that probably becomes ameliorated in late clinical stages of the disease. However, further mechanistic insights on the underlying mechanisms of this effect were not investigated in this study.

The TAB group was initially included in the design of our study as a control group for both ATH and AA patients. Nevertheless, comparisons with the ATH group should be performed with caution, as two out of six patients in the TAB group had a previous history of coronary artery disease (CAD). These patients were free of CAD symptoms and were included in the study since CAD was not considered an exclusion criterion. We are not aware of any study on the role of RL2 in human subjects with CAD. In view of our results on the relationship of serum RL2 in ATH patients with extracranial carotid artery disease and peripheral arterial disease, we believe that relaxin might be related to CAD as well. However, due to the limited number of patients in this group, no safe conclusions can be drawn. Mores studies with CAD patients further subdividing these patients according to the clinical severity of their disease would be appropriate to investigate a potential role of relaxin in CAD.

Our study is not without limitations. First, the small study sample, the fact that the HBD group was not matched with other groups for comorbidities and the heterogeneous pathologies among patient groups, especially in the ATH group, warrant caution when interpreting the results and drawing further conclusions. Second, our results are purely descriptive, with no mechanistic insights on the underlying mechanisms of RL2 in the aforementioned vascular diseases. In order to clarify its potential applicability as a novel therapeutic target, the current literature provides limited data on possible RL2 mechanisms of action in arterial aneurysms. In a recent study, relaxin failed to augment angiotensin II (Ang II)-induced abdominal aortic aneurysms in mice29. The possibilities for the role of RL2 in atherosclerosis include stimulation of vasculogenesis through the upregulation of vascular endothelial growth factor (VEGF) transcript30,31,32,33,34, promotion of vasodilation through NO production35,36,37,38,39,40,41, reduction of systemic arterial resistance and increase of global artery compliance42,43,44,45,46,47, prevention of vascular dysfunction and amelioration of increased responsiveness to Ang II48.

In conclusion, serum RL2 is higher in patients with an arterial aneurysm, showing a positive relationship with the size of the aneurysmatic dilatation. The RL2 level is inversely correlated with the severity of the atherosclerotic disease. Studies with a larger cohort incorporating a consistent study population are warranted to verify our results and shed light on the mechanistic background of these processes.

Author contribution

Konstantinos PAPOUTSIS and Sotirios GEORGOPOULOS designed the study; Konstantinos PAPOUTSIS, Ioannis KARAVOKYROS, and Sotirios GEORGOPOULOS acquired the data; Alkistis KAPELOUZOU analyzed and interpreted data; Konstantinos PAPOUTSIS, Alkistis KAPELOUZOU, Diamantis I TSILIMIGRAS drafed the manuscript; Konstantinos PAPOUTSIS, Alkistis KAPELOUZOU, Diamantis I TSILIMIGRAS, Nikolaos PATELIS, Georgios KOUVELOS, Dimitrios SCHIZAS, Ioannis KARAVOKYROS, Sotirios GEORGOPOULOS made critical revision: Alkistis KAPELOUZOU and Sotirios GEORGOPOULOS supervised the study.