Spontaneous subarachnoid hemorrhage (SAH) has an annual incidence rate of 4–28 cases per 100,000 people.1,2 SAH accounts for about 80% of all nontraumatic extravasated bleeding into the subarachnoid space, 5% of stroke deaths and over a quarter of potential life years lost due to stroke.1,3,4 Approximately 15% of SAH patients die after aneurysmal rupture. Another 25–50% die within a month of the bleeding. Of those who survive, 40% present disabling sequelae.5,6 It is estimated that cerebral vasospasm (CVS) is responsible for neurological deterioration, and even death, in 15–20% of patients with SAH.5,7 CVS is characterized by diffuse and long-lasting arterial constriction. Several vasoconstrictor proteins have been shown to contribute to this narrowing process.8–11 However, Urotensin-II (U-II), defined as the most potent vasoconstrictor peptide in mammals according to Ames et al. research study, is not among these biomarkers.12

U-II is an 11-amino-acid peptide with a cysteine disulfide bond, derived from the polypeptide precursor known as prepro-urotensin-II (preproU-II).13 Proteolytic cleavage of the C-terminal fragment from this precursor is required for biological activity. The result of this proteolysis is an undecapeptide with a cyclic hexapeptide sequence, fundamental for this hormonal action.14 Once the active form of the peptide is generated, U-II mediates its biological action by interacting with a specific plasma membrane G-protein coupled receptor identified as GRP14, or UT.12 This receptor binds different U-II sequences, including the U-II fragment (4–11) and U-II (5–11), as well as the urotensin-related peptide (URP), which derives from a different precursor.15 URP is shorter (octapeptide), but exhibits the cyclic hexapeptide core sequence. U-II expression has been found throughout the heart, kidney, urogenital system and nervous system.16–18 Tian et al. described its expression, as well as UT expression, in the cytoplasm of neurons and in vascular endothelial cells from rats.19 Our research group recently found that high doses of U-II (10μM) in intact rat cerebral arteries evoke arterial contraction. However, in depolarized vascular smooth muscle, lower U-II concentrations (0.1 and 1μM) cause dose-dependent arterial contraction.20 This effect could facilitate arterial CVS in vascular pathophysiological processes such as SAH, where sustained depolarization is present.21

The objective of the present study was to determine the normal range for U-II serum levels and analyze U-II serum and mRNA expression values for the urotensinergic system genes (U-II, URP and UT) in an experimental murine model of SAH; with the purpose of using these molecules as biomarkers of arterial vasoconstriction in SAH.

Procedures were performed in the experimental operating room of the Biomedicine Institute of Seville (Instituto de Biomedicina de Sevilla [IBiS]/CSIC University of Seville), at the Virgen del Rocío University Hospital, Seville, Spain. This research project was overseen and approved by our hospital's Animal Experimentation Committee. It met all the legal requirements and ethical standards for research established by current legislation (Directive 2010/63/EU).

Quantitative variables were presented as medians (interquartile range [IQR]: P25-P75), since they followed a non-normal distribution. We executed the non-parametric Wilcoxon test to analyze within-group differences, and the Mann Whitney U test to verify between-group differences. If significant differences were obtained, 95% confidence intervals (CI) were calculated. Spearman's rank correlation was used to test the association between biomarkers analyzed in this study. Receiver Operator Characteristic (ROC) curve analysis was performed on U-II serum values and on U-II, URP and UT 2−ΔCt values to allocate rats in the Sham or SAH groups. The resulting area under the curve (AUC) was used to establish a cut-off point for animal classification, choosing the point which reached the greatest sum of sensitivity plus specificity. Statistical significance was defined as p<0.05. Statistical analyses were performed using SPSS software (Version 20.0, Chicago, IL, USA).

A total of 96 animals underwent surgery. They were classified as follows: 22 Sham and 74 SAH. Four animals (including one Sham animal) died within 5 days of the surgical intervention.

The macroscopic evaluation of the brains showed that one SAH rat presented an intercisural hematoma.

U-II, URP and UT mRNA expression

For conducting PCR, half lymphocyte samples were randomly selected from both animal groups. The remaining samples were stored for possible future analysis. Thus, we selected 37 SAH samples and 11 Sham samples. Three of the SAH group samples did not have sufficient RNA for inclusion in the analysis.

The results showed that the SAH rats had higher expression levels for URP and UT genes: URP 2−ΔCt=7.07×10−6 (IQR 7.48×10−7–6.40×10−3) SAH vs. URP 2−ΔCt=8.38×10−7 (IQR 4.37×10−7–11.58×10−7) Sham, p=0.037; UT 2−ΔCt=1.97×10−6 (IQR 2.26×10−7–4.49×10−5) SAH vs. UT 2−ΔCt=2.22×10−7 (IQR 1.37×10−7–4.50×10−7) Sham, p=0.046 (Fig. 1). U-II expression levels did not reach statistical significance: U-II 2−ΔCt=1.28×10−7 (IQR 2.84×10−8–2.04×10−6) SAH vs. U-II 2−ΔCt=4.02×10−8 (IQR 2.55×10−8–9.64×10−8) Sham, p=0.175.

Figure 1.

Expression of U-II (A), URP (B) and UT (C) by quantitative RT-PCR assay on the fifth day after brain exposure to blood (SAH) compared to saline injection (Sham). (U-II: p = 0.175; URP: p = 0.037; UT: p = 0.046 relative to Sham).

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In the correlation analysis, in the SAH group, all the quantified gene expressions correlated positively with each other: U-II vs. URP r=0.786, p<0.001; U-II vs. UT r=0.727, p<0.001; URP vs. UT r=0.906, p<0.001. Additionally, URP mRNA showed a positive correlation with U-II serum levels on day 1 (r=0.490; p=0.024) and with U-II serum levels on day 5 (r=0.409; p=0.038). In the Sham group, just correlation between U-II 2−ΔCt and UT 2−ΔCt was found (r=0.767; p=0.016).

Receiver operating characteristics curves

ROC curves were calculated for U-II serum levels and for U-II, URP and UT mRNA expression. The analysis showed that day 5U-II serum levels, URP mRNA and UT mRNA could discriminate between Sham and SAH rats. The AUC for these variables were: AUC=0.691; 95%CI: 0.565–0.817; for day 5U-II serum levels; AUC=0.706; 95%CI: 0.543–0.868; for URP; and, AUC=0.713; 95%CI: 0.540–0.887; for UT (Fig. 2).

Figure 2.

ROC analysis comparing sensitivity to 1-specificity of serum U-II on fifth day (A), URP mRNA (B) and UT mRNA (C) to discriminate SAH rats. (A: AUC=0.691 [95%CI: 0.565–0.817], p=0.008; AUC=0.706 [95%CI: 0.543–0.868] p=0.038; C: AUC=0.713 [95%CI: 0.540–0.887] p=0.046).

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When examining the coordinates of the ROC curve for day 5 U-II serum levels, we observed that the best cut-off, chosen as the one which reached the greatest sum of sensitivity plus specificity, was the value previously defined as the normal median basal serum level for U-II in rats: 0.56pg/mL, with 66.2% sensitivity and 76.2% specificity, 2.15 positive likelihood ratio (LR+), 0.42 negative likelihood ratio (LR−). In our series, the best cut-offs for gene expression were 2−ΔCt=3.77×10−7 for URP, and 2−ΔCt=1.62×10−6 for UT. These cut-offs were applied in our model to determine the accuracy of the variables. URP mRNA displayed 61.3% sensitivity and 83.3% specificity, 3.67 LR+, 0.46 LR−; and UT mRNA showed 60% sensitivity and 80% specificity, 3 LR+, 0.5 LR−.

This is the first study carried out in an experimental murine model to demonstrate that U-II serum levels are significantly elevated on fifth day after SAH, date that coincides with the usual CVS period in both rats24 and humans.25 Moreover, we found that the three urotensinergic system genes were up-regulated after in vivo brain exposure to blood injected into the subarachnoid space. We were also able to define the normal range for U-II serum levels in rats.

The fact that U-II is widely recognized as a peptide with vasoactive properties,26,27 yet no research exists on its role in SAH and its complications, led us to investigate urotensinergic genes in this pathology. In doing so, we detected changes not only in U-II serum level, but in the expression of URP and UT genes as well. We also uncovered the ability of these three genes to discriminate SAH rats. Although no research studies have examined the role of the urotensinergic system in SAH, a recent paper suggested its implication in arterial vasospasm development, based on U-II contraction activity in both intact and depolarized arteries.20 In the present study, the fact that rats with SAH suffer a delayed increase in their gene expression and protein production levels compared to baseline and Sham animals, points to urotensinergic activation in SAH. The increase in plasmatic U-II, limited to the SAH group and confined to the usual CVS period in both rats24 and humans,25 opens the door to a wide range of possibilities for the study of U-II as a biomarker for cerebral arterial vasoconstriction and even the possibility of constituting a new therapeutic target.

The results concerning the correlation between quantified gene expression levels coincides with Yoshimoto et al., who pointed out U-II's function as an autocrine/paracrine.28 Based on our observations, we could highlight this peptide's autocrine activity (correlation between three gene expressions of the urotensinergic system, within the lymphocyte) and its possible paracrine activity (correlation between lymphocyte gene expression, specifically URP, and protein serum levels). Nevertheless, we should note that although there are other sources of U-II release throughout the organism, we only measured mRNA production in the lymphocyte.

U-II has been proposed as a biomarker for various diseases in humans, since its concentration results in a variety of clinical and experimental pathologies, including renal and heart failure, essential and portal hypertension, diabetes, and liver cirrhosis.29 But before using U-II quantification to assess disease onset or progression, we must first establish a normal range for its physiological serum concentration. Several authors have attempted this, and while most agree that U-II serum concentration is found in the picomolar range in species that have been studied, the reports do not agree on the protein's exact concentration range.30–33 On the other hand, some authors suggest that the physiological concentration of U-II is under detectable limits.34 This lack of consensus stems from the absence of interchangeability between the analytical assays employed for U-II quantification.35 These methods include radioimmunoassay, radioreceptor assays, enzyme immunoassays and ELISA.36 The main obstacle is that these methods use antibodies for, but not limited to, U-II (e.g. U-II metabolites). In nonhuman U-II plasma quantification, there is an additional problem: despite the high conservation of this protein throughout the species, the exact sequence varies between them.36 As a result, specific antibodies must be designed for each species. In this study, we solved this problem by using specific biochemical reagents for Rattus norvegicus, in both ELISA and PCR execution. As a result, we were able to define the normal range for U-II serum levels in rats measured by fluorescent enzyme immunoassay (EIA, Phoenix Pharmaceuticals, Inc.). This range of normal values is a significant finding, given that it could serve as a reference for assessing U-II serum levels not only in SAH but in other pathologies as well.

The major strengths of this study rest in the utilization of specific biochemical reagents for the species analysis. Additionally, we performed a very easy and reproducible murine model, which would allow other researchers to reproduce our experiments and continue with this line of investigation. Finally, this animal model of SAH allowed us to study biomarker variation in vivo, and thereby study the progression of the disease in real time.

There were some limitations to the present study. The main limitation was the lack of human analysis. We consider that experimental analysis is important prior to human studies, but we recognize that once demonstrated the up-regulation of the urotensinergic system genes in a SAH model, it is necessary to analyze the same biomarkers in SAH human pathology. We analyzed two blood samples, at baseline and on day 5 after surgery, but did not analyze biomarker evolution throughout the development of the disease. By analyzing more blood samples prior to sacrifice, we could define the kinetics of metabolite production. This research could also benefit from the analysis of other tissues, such as brain tissue, which would provide information on tissue status and their potential gene production.

Ours is the first study carried out in an experimental murine model to demonstrate that U-II protein serum levels, as well as U-II, URP and UT mRNA expression levels are up-regulated on the fifth day after a percutaneous SAH, usual CVS period in both rats24 and humans.25 Furthermore, the three genes showed a high discriminative ability to identify SAH rats. These findings suggest that measurements of gene expression or production, depending on the gene, could serve as biomarkers for SAH onset and progression. Additional studies on SAH patients would be necessary to ascertain the U-II correlation with CVS development, and subsequently determine if this tendency is also observed in humans. These noninvasive analyses could offer physicians early and accurate information on a patient's condition. Furthermore, these results open a new research line for the design of new therapeutic targets for the treatment of the SAH pathology.

This study was funded by a grant from the Instituto de Salud Carlos III (Spanish National Health System) (10/02044), Consejería de Igualdad, Salud y Políticas Sociales de Andalucía, Spain (PI-0136-2012) and Consejería de Igualdad, Salud y Políticas Sociales de Andalucía, Spain (PI-0002-2013).

The manuscript has been read and approved by all of the authors and each author believes that the manuscript represents honest work.

The authors declare no conflicting interest in this work.