Insights into Endothelin-3 and Multiple Sclerosis

Introduction

Endothelin peptides (ETs) comprise of three structurally similar 21-amino acid vasoactive peptides. Endothelin-1 (ET1) and endothelin-2 (ET2) activate two G-protein coupled receptors ETA and ETB, with similar affinity, whereas endothelin-3 (ET3) has a high affinity for the ETB subtype. ET3 is the most abundant ET peptide in the rat brain, mainly localized to neurons and glia of the neostriatum, hypothalamic nuclei, hippocampus, and Purkinje cells of the cerebellum and medulla oblongata [1]. This led to the proposal that ET3 is the “brain” ET [2]. High expression of brain ETB receptors is observed in astrocytes [3]. Expression of astrocytic ETB receptors is upregulated after brain injury and is known to be a potent mitogen of astrocytes [4]. Furthermore, astrocytes produce and release various bioactive substances, for example, neurotrophic factors, cytokines, chemokines, nitric oxide, and vascular permeability factors, through which they interact with neurons and brain microvessels [4]. Production of astrocyte-derived molecules is stimulated by the activation of astrocytic ETB receptors [5].

The biosynthetic pathway of ETs is considered to be similar: 1. a large precursor designated preproET is formed; 2. after enzymatic removal of a signal peptide, proET is cleaved by a protease leading to the formation of big ET, finally 3. big ET is hydrolyzed into the small, mature ET1 by an endothelin-converting enzyme (ECE). At present, the mammalian ECE family consists of three known forms (ECE-1, ECE-2, ECE-3), which are classified into the neutral endopeptidase group of proteins [6]. Each member of this family shows homology to the others, mainly in the C-terminal part of the sequence that is responsible for catalytic activity. In addition, there are structural similarities in that all neutral endopeptidase members, for example, ECE-1, Kell glycoprotein can proteolytically cleave ET1, ET2, and ET3, with a marked preference for ET3 [7].

While the primary source of circulating ET1 is the vascular endothelial cells [8], these cells do not synthesize ET3. One possible source of circulating big ET3 could be the adrenal gland [2]. Although this peptide has been visualized in secretory cells of the medulla using selective antisera, mature ET3 could not be detected [9,10]. Changes associated with the disease have not been extensively investigated, but concentrations of ET3 increased significantly in hemodialysis patients (together

with big ET1 and big ET2) but with only a moderate rise in corresponding active peptides [11]. If released, further processing of big ET3 could occur within the vasculature by smooth muscle cell ECE [12,13]. Interestingly, the pituitary was observed to have higher levels of ET3 than ET1. Many studies have identified dysregulation of the hypothalamic-pituitary-adrenal axis in patients with MS [14,15]. Indeed, pituitary-ovary axis and ovarian reserve have been investigated, and lower corticotropin like intermediate lobe peptide has been demonstrated. Generally, the hypothalamic-pituitary-adrenal axis has been demonstrated to be hyperactive in MS patients, and putative involvement of plasmatic active ET3 and hypothalamic-pituitary-adrenal axis dysregulation could be hypothesized [15].

This study aims to investigate the possible differences between ET3 plasma levels between MS patients and healthy subjects. Furthermore, we investigate the potential relationship between circulating vasoactive ET3 and cerebral circulation time and the relationship between circulating ET3 and ET1 in the same MS patient cohort.

Materials and Methods

Pregnant or nursing women, heavy smokers, and patients within 30 days of previous therapy were excluded from the study.

A total of 43 patients with proven MS have been included. Blood samples with hemolysis were discarded, and a total of 29 MS patients were considered. The 29 MS patients (11 men and 18 women) had the relapsing-remitting form of MS.

A group of 37 age and sex-matched healthy control subjects were included in the study. Seven subjects were excluded due blood sample hemolysis; therefore a total of 30 healthy subjects (11 men and 19 women) were considered.

In 29 MS patients, peripheral blood samples were collected, and ET3 and ET1 titration were quantified. Peripheral blood samples were collected after 30 minutes of rest in a supine position from the brachial vein and circulating ET3 and ET1 levels were measured. Cerebral circulation time was calculated in all MS patients recruited by digital subtracted angiography (DSA) [16].

Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by ethical board of the AOUS General Hospital Santa Maria alle Scotte, Siena.

Informed consent: Informed consent has been obtained from all individuals included in this study

Results and Discussion

Despite extensive research, there remains a critical gap in our understanding of the mechanisms that promote brain hypoperfusion in MS patients. The most reliable hypothesis in the literature is a vascular dysregulation resulting in increased brain vessel resistance mediated by ET1. However, although ET1 plasma levels are increased in MS with respect to healthy subjects, the relationship between ET1 and cerebral circulation time values remains elusive [17,18]. Here we focus on the ET3 vasoactive peptide, for which little is known. This study has demonstrated that in MS patients the ET3 plasma levels are higher than in healthy controls (Fig.1), but we found no correlation between ET3 levels and cerebral circulation time (Fig.2b). ET3 is positively correlated with ET1 plasma levels (Fig.2d), and both peptides correlate with disease duration (Fig.2a; Fig.2c).

Figure 1

Endothelin-3 (ET3) plasma levels determined from healthy (control) subjects and multiple sclerosis patients (mean ± standard deviation). Statistical significance was demonstrated using Mann-Whitney (two-tailed) *p = 0.0329.

Figure 2

Analysis of correlation among the variables cerebral circulation time (CCT), endothelin-1 (ET1), endothelin-3 (ET3), and disease duration in MS patients. (a) Correlation between ET3 and disease duration. ET3 changes during the disease duration reached significant statistical difference using the Pearson correlation coefficient (two-tailed) ***p = 0,0002. (b) Correlation between ET3 and CCT. The CCT delay was not statistically significantly correlated to increased ET3 titration. (c) Correlation between ET1 and disease duration. ET1 was statistically significantly correlated with disease duration using the Pearson correlation coefficient (two-tailed) r = 0.54025 with a 95% confidence interval between 0.19336 - 0.76708, R2 = 0.29187, **p = 0.0044 (alpha = 0.05). (d) Correlation between circulating ET3 and ET1. Increasing ET3 and ET1 plasmatic titration levels were statistically significantly correlated using Pearson correlation coefficient. (r=0,54025 with 95% confidence interval 0.19336 to 0.76708 and R squared=0,29187). The increasing ET3 plasmatic values are statistically correlated with increasing ET1 plasmatic values and the P (two-tailed) value results as 0,0044 (**)