Clazosentan Affects Temporal Profile of Endothelin-A Expression in Macro- and Microcirculation after Subarachnoid Hemorrhage

Abstract

Subarachnoid hemorrhage is a life-threatening cerebrovascular event, and cerebral vasospasm remains a major cause of poor neurological outcomes. Clazosentan, an endothelin-A receptor antagonist, has been recently approved in Japan to reduce post-subarachnoid hemorrhage vasospasm; however, recurrent vasospasm after cessation of therapy has occasionally been reported, which underlying mechanisms remain unclear. Moreover, endothelin-A receptor is also expressed on pericytes; however, the effects of clazosentan on microvascular endothelin-A receptor remain unexplored. In this study, we employed a rat subarachnoid hemorrhage model to investigate the temporal dynamics of vasospasm and endothelin-A receptor expression in both large arteries and microvessels, and to evaluate the effects of clazosentan administration. Sprague-Dawley rats were assigned to naïve controls, subarachnoid hemorrhage with saline, or subarachnoid hemorrhage with continuous clazosentan administration for 7 days via osmotic pumps. Vasospasm was assessed by arterial wall thickness, and endothelin-A receptor expression was quantified using immunohistochemistry and immunofluorescence, including staining with α-SMA, CD31, and PDGF-β. Clazosentan significantly attenuated vasospasm in the middle and anterior cerebral arteries, and recurrent vasospasm was observed 3 days after cessation of clazosentan, coinciding with sustained upregulation of endothelin-A receptor in these vessels. In microvessels, pericyte density transiently decreased, peaking at a nadir on day 3 post-subarachnoid hemorrhage, while endothelin-A receptor expression on pericytes was highest at the same time point, and further elevated by clazosentan treatment. These findings indicate that clazosentan induces endothelin-A receptor upregulation in both macro- and microcirculation, potentially contributing to recurrent vasospasm after treatment, and highlight the critical role of pericytes in post-subarachnoid hemorrhage vascular regulation.

Introduction

Subarachnoid hemorrhage (SAH), which accounts for approximately 10% of all stroke cases, is typically caused by the rupture of a cerebral aneurysm.¹⁾ Despite substantial advances in medical management, the prognosis for patients with SAH has remained largely unchanged for decades, highlighting the urgent need for the development of innovative therapeutic approaches.²⁾ One of the most severe complications of SAH is cerebral vasospasm, which is angiographically observed in approximately 70% of patients. This condition subsequently leads to delayed cerebral ischemia (DCI) in 20%-40% of cases, significantly impacting patients' functional outcomes and quality of life.³⁾

Clazosentan, an endothelin-A receptor (ET_A-R) antagonist, was clinically approved in Japan in 2022 for the treatment of vasospasm following SAH. It exerts potent vasodilatory effects by reversibly inhibiting the binding of endothelin-1 (ET-1) to ET_A-R expressed on cerebral vascular smooth muscle cells. Clinical studies have demonstrated that clazosentan significantly reduces the combined incidence of vasospasm-related morbidity and all-cause mortality in SAH patients following surgical clipping and coil embolization.^4-7) Administration of clazosentan is approved for up to 14 days post-SAH, coinciding with the typical resolution period of vasospasm. Nonetheless, sporadic cases of vasospasm recurrence beyond 14 days following completion of the standard clazosentan regimen―thereafter referred to as recurrent vasospasm―have been reported.^8,9) This phenomenon, rarely observed with conventional pre-clazosentan therapies,¹⁰⁾ remains poorly understood, and the precise mechanisms involved are not yet fully elucidated. Furthermore, recent studies have indicated that pericytes, which are integral components of the neurovascular unit in microvessels, also express ET_A-R on their cell surface. Pericytes play a pivotal role in regulating microcirculatory dynamics and inflammatory responses, which may contribute to the secondary brain injury after SAH. However, the impact of clazosentan on ET_A-R expression in pericytes remains unexplored.

Accordingly, this study aims to investigate two previously uncharacterized aspects: the molecular mechanisms underlying recurrent vasospasm in major cerebral arteries following clazosentan treatment, and the temporal expression pattern of ET_A-R within the cerebral microcirculation.

Methods

Animal protocols were approved by the Animal Studies Ethics Committee of the Hokkaido University Graduate School of Medicine (approval number: 24-0077). All experimental procedures were conducted following the Institutional Guidelines for Animal Experimentation and the Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan. All animals were housed in standard polypropylene cages (typically 2-3 rats per cage) under specific pathogen-free conditions. The animal facility was maintained at a temperature of 22 ± 2°C, with relative humidity of 50%-60% and a 12-hour light/dark cycle. Rats had ad libitum access to autoclaved food and water. Animals were monitored daily for health and welfare. Consent was not required because this study involved no human subject.

SAH model and clazosentan administration

Male Sprague-Dawley rats (CLEA Japan, Japan), aged 8 weeks and weighing between 250 and 320 g, were used to establish the experimental SAH model, as previously described with minor modifications.¹¹⁾ Briefly, the rats were anesthetized by isoflurane gas anesthesia and then positioned prone in a stereotactic frame. After making a midline scalp incision, a 1-mm burr hole was drilled 5 mm anterior to the bregma, 2 mm to the right of the midline. Fresh autologous blood (300 μL) was drawn from the middle tail artery using a heparin-coated syringe and infused into the prechiasmatic cistern over 30 sec via a 21-gauge needle (Terumo, Japan) angled at 30° in the sagittal plane. The animals were then randomly assigned to one of two groups: the clazosentan-treated group (SAH-CLZ group) and the saline-treated group (SAH-S group). An osmotic pump (2ML1, Alzet, Cupertino, CA, USA) was subcutaneously implanted on the back of the animal, with its catheter inserted into the right external jugular vein for continuous drug administration. Clazosentan was diluted in saline, and a dose of 1 mg/kg/hr of clazosentan solution or saline was administered for up to 7 days, and the animal was sacrificed various time points for histological evaluation.¹²⁾ Animal without SAH induction served as the healthy control group (naïve group).

Immunohistochemistry

At the time of sacrifice, rats were deeply anesthetized and transcardially perfused with saline followed by 4% paraformaldehyde (PFA). The brains were then extracted, fixed in 4% PFA for 24 hours, embedded in paraffin, and sectioned into 4-μm-thick coronal slices, as previously described.¹¹⁾

Vasospasm was evaluated by measuring arterial wall thickness, as outlined in previous studies.¹³⁾ Briefly, following standard hematoxylin and eosin (H&E) staining, the bregma level of the left middle cerebral artery (MCA) and anterior cerebral artery (ACA), corresponding to the contralateral side of the blood injection, was selected for further analysis. The thickness of the arterial wall, including the intima and media, was measured parallel to the shortest axis diameter of the MCA or ACA using ImageJ software. Subsequently, the average thickness of both sides of the same artery was calculated to determine the overall arterial thickness.

ET_A-R expression on the MCA and ACA was analyzed according to a previously described method with minor modifications.¹⁴⁾ Anti-ET_A-R antibody (1:150, GTX21920, GeneTex, Irvine, CA, USA, incubated for 48 hours at room temperature) was used as primary antibody, and Alexa Fluor 647 (1:1000, ab150179, Abcam, Cambridge, MA, USA) was used for secondary antibody (incubated for 1 hour at room temperature). Subsequently, 4′,6-diamidino-2-phenylindole was employed as a fluorescent nuclear counterstain and mounting medium. After staining, the area of the target artery was manually selected using the BZ-X Analyzer (Keyence Co., Osaka, Japan), and the intra-vessel normalized intensity was measured to quantify ET_A-R expression. This value was derived by dividing the signal intensity by the reference intensity as previously reported.^15-17)

To verify the localization of ET_A-R expression on the cerebral artery, double immunofluorescence staining was performed on MCA. Sections were incubated with an anti-ET_A-R antibody, as previously described, and an anti-α-SMA antibody (1:100, ab7817, Abcam, Cambridge, UK) for 1 hour at room temperature. This was followed by incubation with secondary antibodies conjugated to Alexa Fluor 647 and 488 (Abcam, USA).

Microvessel pericyte densities were assessed using anti-COL4 antibody (1:100, ab6586, Abcam), followed by 3,3′-diaminobenzidine chromogen staining, as previously reported.^18,19) After staining the basement membrane with anti-COL4 antibody, six non-overlapping regions of interest (ROIs) were designated in the hippocampus and striatum (bregma: −4 mm) on the contralateral side of the hemorrhage. Nucleated pericyte cell bodies, characteristically recognized by protrusions or “bump and crescent” shapes, were counted, and pericyte density was calculated by dividing the number of pericytes by the total capillary length for each subject. ET_A-R localization and expression on microvessels were assessed as previously described.¹⁴⁾ Tissue sections were incubated at room temperature for 48 hours with anti-PDGF Receptor-β (1:50, 3169S, Cell Signaling, USA), anti-CD31 (1:100, AF3628, R&D Systems, USA), and anti-ET_A-R (1:150, GTX21920, GeneTex, USA). Following primary antibody incubation, secondary antibodies conjugated to Alexa Fluor 568, 488, and 647 were applied for 1 hour at room temperature. Microvessels with minor axis of 9-39 μm in the hippocampus and striatum were selected for further analysis. Intra-vascular inflorescence of ET_A-R intensity was auto-measured as previously described. Super-resolution images were further acquired to verify the location of ET_A-R on microvessel using an Eclipse Ti2e microscope (Nikon, Tokyo, Japan) equipped with a PlanApo 20×/0.8 dry objective lens, a Ti2-S-SE motorized stage, a Ti2-CTRE microscope controller, and DeepSIM (CrestOptics, Rome, Italy) as previously reported.^20,21) The detector used in this study was a PRIME BSI scientific complementary metal-oxide semiconductor camera (Teledyne Photometrics, Tucson, AZ, USA). The cells were illuminated with a CELESTA laser light engines (Lumencor, NW Greenbrier Parkway, Beaverton, OR, USA) through an FF01-391/477/549/639/741 excitation filter (Semrock, Rochester, NY, USA). Emission filters were selected as follows: FF01-511/20 (Semrock) for Alexa Fluor 488, FF01-595/31 (Semrock) for Alexa Fluor 568, and ET720/60m (Chroma Technology Corp., Bellows Falls, VT, USA) for Alexa Fluor 647. An FF421/491/567/659/776-Di01 dichroic mirror (Semrock) was used throughout this analysis. Control of the microscopes and peripheral equipment was managed by using VisiView Ver6 (Visitron System GmbH, Puchheim, Germany).

Statistics analyses

Data were expressed as the mean ± standard error. Statistical analyses were performed using JMP Pro 14 software (SAS Institute Inc., NC, USA). The sample size was chosen based on comparable experiments from our previous experiments.¹¹⁾ Statistical comparisons between two different groups were made using the t-test, the differences between three groups were examined using a one-factor analysis of variance followed by Tukey's Honestly Significant Difference test or Student's t-test. Probability values of p < 0.05 were considered statistically significant.

Results

Effect of clazosentan in large vessels

The temporal course of vasospasm was initially assessed in the rat SAH model. Arterial thickening, a hallmark of vasospasm, was observed to emerge on day 3 and persist until day 7 post-SAH in both the MCA and ACA (Supplementary Figure 1). Based on these observations and 7-day continuous infusion period of the Alzet pump, day 7 are selected as major time point for vascular analysis, with day 3 included as an additional reference. Evaluation of the inhibitory effect of clazosentan revealed that its administration significantly attenuated vasospasm at day 7 compared with the saline-treated group in both the MCA (Figure 1A) and ACA (Supplementary Figure 2A), with a similar reduction observed at day 3 in both vessels (Supplementary Figure 3). To assess recurrent vasospasm, three additional time points were evaluated in animals receiving clazosentan: the day of clazosentan cessation (day 7), 3 days post-cessation (day 7 + 3), and 7 days post-cessation (day 7 + 7). Notably, vasospasm significantly recurred at day 7 + 3 compared with day 7 in the MCA, and returned to baseline levels by day 7 + 7 (Figure 1B). Although not statistically significant, a similar trend was observed in the ACA (Supplementary Figure 2B). ET_A-R expression following clazosentan administration was further investigated to elucidate the mechanisms underlying recurrent vasospasm. At day 7, clazosentan treatment significantly increased ET_A-R expression compared with the SAH-S group in both the MCA (Figure 2A) and ACA (Supplementary Figure 4A). At a later time point (14 days post-SAH), the clazosentan group (day 7+7) continued to exhibit elevated ET_A-R expression compared with the saline group (day 14) in both the MCA (Figure 2B) and ACA (Supplementary Figure 4B). The cellular localization of ET_A-R was evaluated by double immunostaining with smooth muscle cell marker α-SMA, revealing predominant expression on the luminal surface of vascular smooth muscle cells (Figure 2C). These findings indicate that clazosentan induces overexpression of ET_A-R via a negative feedback mechanism, which may contribute to the observed recurrent vasospasm.

Figure 1.

(A) Vasospasm of MCA following SAH was evaluated at day 7 by hematoxylin and eosin (H&E) staining. The SAH with saline injection (SAH-S) exhibited increased vasospasm, while the SAH with clazosentan group demonstrated significant reduction of vasospasm.

(B) Recurrent vasospasm was evaluated by H&E staining. Representative images of MCA at day 7 (immediately after clazosentan cessation), day 7 + 3 (3 days after cessation of clazosentan), and day 7 + 7 (7 days after cessation of clazosentan). Arterial thickening was significantly elevated 3 days after cessation of clazosentan compared with timing of cessation (day 7), indicative of recurrent vasospasm. The recurrent vasospasm was then alleviated 7 days after cessation of clazosentan (day 7+7) (*p < 0.05, **p < 0.01). The size bar = 50 mm.

MCA: middle cerebral artery; SAH: subarachnoid hemorrhage

Figure 2.

(A) ET_A-R expression of MCA following SAH was evaluated at day 7. The SAH with clazosentan injection (SAH-CLZ) exhibited increased ET_A-R expression, while the SAH with saline group showed similar results with naïve control.

(B) ET_A-R expression of MCA following SAH was evaluated at later time point (14 days from SAH). ET_A-R expression of clazosentan-group showed that is still significantly higher 7 days after cessation of clazosentan (day 7 + 7) compared with that of saline treated group (day 14).

(C) The cellular localization of ET_A-R (red) was evaluated by double immunostaining with smooth muscle cell marker α-SMA (green). ET_A-R was predominantly expressed on the luminal surface of vascular smooth muscle cells. (*p < 0.05, **p < 0.01). The size bar = 50 mm.

α-SMA: α-smooth muscle actin; ET_A-R: endothelin-A receptor; MCA: middle cerebral artery; SAH: subarachnoid hemorrhage

Effect of clazosentan on microvessels

Temporal profile of pericyte expression in microvessels after SAH was evaluated. Pericyte density in hippocampus showed a significant change, which pericyte density gradually decreased and reached lowest value at 3 days after SAH, and then recovered to the normal level at day 7 (Figure 3A). A similar trend was observed in the striatum; however, the changes were more subtle and did not reach statistical significance (p = 0.11). Interestingly, ET_A-R expression exhibited an inverse pattern, with levels increasing after SAH, peaking at day 3, and returning to baseline by day 7 in both the hippocampus and striatum (Figure 3B). The spatial distribution of ET_A-R expression was further examined in naïve rats and rats at day 3 post-SAH. In naïve animals, ET_A-R expression was predominantly colocalized with endothelial cells (CD31), whereas in day 3 SAH rats, ET_A-R was detected in both endothelial cells and pericytes (PDGF-β) (Figure 4). The temporal profile of ET_A-R expression under clazosentan treatment was also assessed. The SAH-CLZ group exhibited higher ET_A-R expression at day 7 compared with both naïve animals and the SAH-S group, with the effect more pronounced in the striatum than in the hippocampus (Figure 5). These findings indicate that, under physiological conditions, ET_A-R is primarily expressed on endothelial cells. Following SAH, pericytes begin to express ET_A-R, and clazosentan further upregulates its expression within the cerebral microcirculation.

Figure 3.

(A) Pericyte density is evaluated by anti-COL4 immunohistochemistry staining for hippocampus and striatum. Pericyte density campus gradually decreased and reached lowest value at 3 days after SAH, and then recovered to the normal level at day 7, where black arrows indicate pericyte.

(B) The temporal expression profiles of ET_A-R (green) on pericytes (PDGFR-β, red) were evaluated in the microcirculation following SAH in the hippocampus and striatum. Expression of ET_A-R showed a gradual increase, peaking at day 3, and then returning to baseline levels by day 7 (*p < 0.05, **p < 0.01). The size bar = 50 mm.

ET_A-R: endothelin-A receptor; PDGFR-β: Platelet-derived growth factor-β; SAH: subarachnoid hemorrhage

Figure 4.

Super-resolution images for verifying the location of ET_A-R on microvessels. The locations of the ET_A-R (green), endothelial cell (CD31, blue), and pericyte (PDGF-β, red) expression inside the arterial wall were analyzed by its normalized intensity. While ET_A-R mainly colocalize with endothelium cells in the naïve animal (upper raw), ET_A-R was expressed on both endothelium cells and pericyte 3 days after SAH (lower raw). Graph in the right side represents the special distribution between A and B in the merged figure. The size bar = 1 mm.

ET_A-R: endothelin-A receptor; PDGF-β: Platelet-derived growth factor-β; SAH: subarachnoid hemorrhage

Figure 5.

ET_A-R expression on pericyte was evaluated with naïve, SAH-saline, and SAH-clazosentan group at day 7. The SAH-clazosentan group exhibited increased ET_A-R expression trend, while the SAH with saline group showed similar results with naïve group (*p < 0.05, **p < 0.01). The size bar = 50 mm.

ET_A-R: endothelin-A receptor; SAH: subarachnoid hemorrhage

Discussion

In this study, we demonstrated that clazosentan effectively attenuated vasospasm in the large arteries, while concurrently inducing upregulation of ET_A-R in both macro- and microcirculation. These findings may provide an explanation for the recurrent vasospasm observed following clazosentan treatment and underscore the need for further experiments aimed at improving the microcirculatory environment.

Clinically, angiographic vasospasm typically begins around days 3 to 4 after aneurysm rupture, peaks between days 7 and 10, and resolves by day 14.¹⁰⁾ Although the precise mechanisms remain incompletely understood, ET-1 is considered a key factor, as it binds to ET_A-R expressed on smooth muscle cells of cerebral arteries, thereby contributing to the inhibition of severe vasoconstriction. Numerous studies have shown that clazosentan reduces vasospasm;^22,23) however, reports regarding the temporal expression of ET_A-R following SAH remain limited. Vatter et al. reported that immunohistochemical staining of the smooth muscle layer did not show a significant increase in ET_A-R expression following SAH.²⁴⁾ Similarly, Povlsen et al. observed a temporal increase in ET_B-R expression after SAH, which peaked at day 3 and returned to baseline by day 4.²⁵⁾ These findings align with our observation that SAH alone does not solely upregulate ET_A-R at 7 days post-SAH. Therefore, the upregulation of ET_A-R in the clazosentan treatment group observed in our study is likely due to clazosentan's high affinity, which induces negative feedback on receptor expression. Since widespread clinical use of clazosentan has just been started, and recurrent vasospasm following clazosentan treatment has only recently been recognized, further evaluation of this phenomenon is highly warranted. Currently, there are no studies investigating the mechanisms underlying the recurrent vasospasm after cessation of clazosentan. According to our results, we not only observed that recurrent vasospasm after cessation of clazosentan in animal models, but also found ET_A-R expression in large cerebral arteries remained elevated even 7 days after clazosentan withdrawal. Taken together, this mechanism may represent one of the contributors to the recurrent vasospasm observed clinically.

We also focused on ET_A-R expression on pericyte in this study. The neurovascular unit (NVU) is the fundamental structure that maintains the blood-brain barrier, regulates cerebral blood flow, and participates inflammatory responses. Recent reports have highlighted the critical role of pericytes in regulating NVU functions.^26,27) The kinetic changes in pericytes following SAH have yielded divergent results. Schwarting et al. reported no significant difference in pericyte density 24 hours after SAH induction compared with sham surgery.²⁸⁾ In contrast, other studies have observed a significant decrease in pericyte density between 24 and 48 hours following SAH.^29,30) We hypothesize that these discrepancies arise from differences in the time points of observation. In our experiment, a trend of pericyte loss was noted at day 1, and the number of pericytes subsequently decreased to its lowest level at day 3. Interestingly, by day 7, the number of pericytes had returned to baseline levels, indicating a rapid recovery of pericytes following SAH induction. Pericyte recovery has been reported in other studies that pericyte density decreased after 10 consecutive days of sleep deprivation, but was completely restored within 21 days. Notably, pericyte loss was not attributed to apoptotic activity, suggesting the possibility of a rapid turnover of pericytes in the brain.³¹⁾ In this study, we also observed that ET_A-R expression on pericyte was upregulated in the SAH-CLZ group compared with other groups. To the best of our knowledge, this is the first article focusing on this phenomenon. However, the precise role of ET_A-R upregulation of pericyte, as well as the overall role or pericyte after SAH, remains to be elucidated. Previous studies have reported conflicting results, with some suggesting that pericytes are not involved in the microcirculation after SAH,³²⁾ while another has reported that pericyte loss following SAH induces microthrombus formation and low perfusion.²⁹⁾ Other functions of pericytes after SAH have also been described, including the induction of capillary spasm and low perfusion through a calcium-dependent mechanism,³³⁾ with calcium channel blockers, such as nimodipine, reducing microvasospasm.²⁸⁾ Furthermore, pericytes are known to regulate the differentiation and maturation of oligodendrocytes by releasing tissue inhibitors of matrix metalloproteinase-3 (TIMPS), and SAH has been shown to decrease TIMPS expression.³⁴⁾ These findings indicate the complex, multifaceted role of pericytes in the brain, highlighting the need for further investigation into the role of ET_A-R in these processes.

Several limitations of this study should be acknowledged. First, ET_A-R expression was assessed solely through immunohistochemistry. Protein analysis, such as Western blotting, could add further value; however, it would not be able to distinguish the different expression patterns between large and small vessels. Second, while clazosentan is typically administered for 14 days in clinical settings, our study only utilized a 7-day treatment regimen. Thus, the changes in ET_A-R expression between days 7 and 14 remain unclear. Third, the functional analysis of ET_A-R on microcirculation has not been evaluated in this study. Since pericyte possesses multiple function and further evaluation is necessary.

Conclusion

Continuous clazosentan administration in the SAH model increased the expression of ET_A-R in both macro- and microcirculation, which may contribute to the recurrent vasospasm observed following clazosentan treatment.

Disclamer

Author Miki Fujimura is one of the Editorial Board members of the Journal. This author was not involved in the peer-review or decision-making process for this paper.

Author Contributions

MK designed the experiment, YYZ wrote the first draft, and conducted the statistical analysis; YYZ, SG, YF, EY, YN, ZL, RT, TI, and TH conducted the experimental procedures; YO and MF critically revised the article. Masahito Kawabori/MK acted as guarantor.

Conflicts of Interest

Fujimura M. received a research grant and serves as a consultant to Nxera Pharma. Other authors have no conflict of interest to disclose.

Consent for Publication

All authors agreed to submit the manuscript, read and approved the final draft and take full responsibility of its content, including the accuracy of the data.

Availability of Supporting Data

All additional files are included in the manuscript. The data that support the findings of this study are available from the corresponding author, [MK], upon reasonable request.

Supplementary Material

Supplementary Figure 1

Temporal profile of the vasospasm following SAH is evaluated on anterior cerebral artery (ACA) and middle cerebral artery (MCA). Arterial thickening, a hallmark of vasospasm begins on day 3 and persist until day 7 post-SAH.

SAH: subarachnoid hemorrhage.

Supplementary Figure 2

(A) Vasospasm of ACA following SAH was evaluated at day 7. The SAH with saline injection (SAH-S) exhibited significantly severe vasospasm, while the SAH with clazosentan group demonstrated significant reduction in vasospasm compared with SAH-S group (*p < 0.05). The size bar = 50 mm.

(B) Recurrent vasospasm was evaluated at ACA. Arterial thickening was elevated 3 days after cessation of clazosentan (SAH-CLZ day 7 + 3) compared with timing of cessation (day 7); however, no statistical difference was seen between the groups. The size bar = 50 mm.

ACA: anterior cerebral artery; SAH: subarachnoid hemorrhage.

Supplementary Figure 3

Vasospasm of ACA following SAH was evaluated at day 3. The SAH with saline injection (SAH-S) exhibited increased vasospasm, while the SAH with clazosentan group demonstrated significant reduction of vasospasm (**p < 0.01, ***p < 0.001) The size bar = 50 mm.

ACA: anterior cerebral artery; SAH: subarachnoid hemorrhage.

Supplementary Figure 4

(A) ET_A-R expression of ACA following SAH was evaluated at day 7. The SAH with clazosentan injection (SAH-CLZ) exhibited increased ET_A-R expression, while the SAH with saline group showed similar results with naïve control.

(B) ET_A-R expression of ACA following SAH was evaluated at later time point (14 days from SAH). ET_A-R expression of clazosentan-group showed that is still significantly higher 7 days after cessation of clazosentan (day 7+7) compared with that of saline treated group (day 14).

(**p < 0.01, ***p < 0.001) The size bar = 50 mm.

ACA: anterior cerebral artery; ET_A-R: endothelin-A receptor; SAH: subarachnoid hemorrhage.

Funding Statement

This study was funded by the Japan Society for the Promotion of Science KAKENHI (Grant Number 23K08535), and this research was partially supported by Nxera Pharma.