Bradykinin‐bradykinin receptor (B1R) signalling is involved in the blood–brain barrier disruption in moyamoya disease

Abstract Moyamoya disease (MMD) is a rare disorder of the cerebrovascular system. It is a steno‐occlusive disease that involves angiogenesis and blood–brain barrier (BBB) disruption. Bradykinin (BK), its metabolite des‐Arg9‐BK, and receptor (B1R) affect angiogenesis and BBB integrity. In this study, we aimed to investigate the changes in BK, B1R and des‐Arg9‐BK levels in the serum and brain tissues of patients with MMD and explore the underlying mechanism of these markers in MMD. We obtained the serum samples and superficial temporal artery (STA) tissue of patients with MMD from the Department of Neurosurgery of the Jining First People's Hospital. First, we measured BK, des‐Arg9‐BK and B1R levels in the serum of patients by means of ELISA. Next, we performed immunofluorescence to determine B1R expression in STA tissues. Finally, we determined the underlying mechanism through Western blot, angiogenesis assay, immunofluorescence, transendothelial electrical resistance and transcytosis assays. Our results demonstrated a significant increase in the BK, des‐Arg9‐BK and B1R levels in the serum of patients with MMD compared to healthy controls. Furthermore, an increase in the B1R expression level was observed in the STA tissues of patients with MMD. BK and des‐Arg9‐BK could promote the migratory and proliferative abilities of bEnd.3 cells and inhibited the formation of bEnd.3 cell tubes. In vitro BBB model showed that BK and des‐Arg9‐BK could reduce claudin‐5, ZO‐1 and occluding expression and BBB disruption. To the best of our knowledge, our results show an increase in BK and B1R levels in the serum and STA tissues of patients with MMD. BK and Des‐Arg9‐BK could inhibit angiogenesis, promote migratory and proliferative capacities of cells, and disrupt BBB integrity. Therefore, regulating BK, des‐Arg9‐BK and B1R levels in the serum and the brain could be potential strategies for treating patients with MMD.


| BACKG ROU N D
Moyamoya disease (MMD) is a rare disorder of the cerebrovascular system.Progressive narrowing or occlusion of the intracranial internal carotid arteries and their branches are characteristics of MMD.This leads to the formation of moyamoya vessels, which are fragile collateral blood vessels at the base of the brain. 1,2The moyamoya vessel means 'puff of smoke' in Japanese.These vessels lead to chronic ischemia, haemorrhagic stroke, transient ischemic attack and cognitive impairment.If left untreated, these moyamoya vessels could cause permanent neurological deficits and disabilities.
MMD is highly prevalent in Asian populations, specifically in East Asian populations (China, Japan, Korea, etc.).The prevalence of MMD is 0.35-0.54/100,000individuals per year. 3Genetic predisposition, environmental factors and immune factors are associated with MMD pathogenesis; however, the aetiology and pathogenesis of MMD are unclear.
Angiogenesis is the formation of new blood vessels from preexisting ones, which is crucially involved in the development and maintenance of the vascular network in the brain. 4,5Angiogenesis orchestrates the proliferation, differentiation and migration of endothelial cells (ECs) and pericytes.The migration and invasion of cells are essential steps in angiogenesis as ECs migrate toward the site of angiogenesis and invade the surrounding tissue to form new blood vessels.In addition to physiological angiogenesis, abnormal or pathological angiogenesis has been implicated in various neurological disorders, including MMD.Recent studies have shown that the dysregulation of angiogenic factors, such as vascular endothelial growth factor (VEGF), angiopoietin-1, transforming growth factorbeta 1 and platelet-derived growth factor could cause abnormal angiogenesis in patients with MMD. 6,7These angiogenic factors stimulate the proliferative and migratory abilities of ECs and pericytes and promote the production of extracellular matrix (ECM) proteins and new blood vessel formation.However, the underlying mechanisms of the dysregulation of these angiogenic factors and the aberrant angiogenesis in patients with MMD are poorly understood.
Interestingly, studies have shown the involvement of bradykinin (BK) metabolism in angiogenesis and cell migration 8,9 ; thus, BK could be involved in MMD pathogenesis.
BK is a nine-amino acid peptide found in several cells and tissues involved in various physiological and pathological processes.BK binds to B1 and B2 (B1R and B2R), G-protein-coupled receptors, widely expressed by different cell types to exert its effects.B1R is primarily expressed under inflammatory conditions, whereas B2R is constitutively expressed. 10,11BK activates ECs to increase VEGF expression, thereby promoting the migration and proliferation of ECs. 12 Additionally, BK activates smooth muscle cells and fibroblasts to secrete VEGF and matrix metalloproteinases (MMPs), which facilitate the remodelling and maturation of vessels.Des-Arg9-BK is a BK metabolite that impacts biological processes, such as angiogenesis and cell migration. 13BK is cleaved by the angiotensin-converting enzyme or aminopeptidases to generate des-Arg9-BK.Furthermore, des-Arg9-BK has a higher affinity to the B1R compared to B2R and is primarily involved in inflammatory conditions. 14Recent studies have shown that BK and des-Arg9-BK are involved in angiogenesis and cell migration.BK enhances the proliferative and migratory ability of ECs and stimulates new blood vessel formation in vivo.Des-Arg9-BK enhances angiogenesis under inflammatory conditions and the migration as well as invasion of cancer cells. 15,16reover, MMD affects the blood-brain barrier (BBB), a complex network of specialized cells essential for maintaining a stable environment for the brain.BBB protects the brain from harmful substances in the blood. 17BBB disruption occurs in patients with MMD, which increases the permeability and susceptibility to neurotoxic insults.The disruption of BBB facilitates the proinflammatory cytokines, immune cells and factors to enter the brain, which damages the brain and contributes to MMD pathogenesis.Studies have explored the role and mechanism of the BK/B1R in BBB, which revealed that BK could cause injury to BBB.Interestingly, the knockdown of B1R could exert beneficial effects. 18,19ltiple studies have explored the mechanism of MMD pathogenesis; however, no study has investigated the correlation between BK and MMD.Therefore, in this study, we aimed to investigate BK, B1R and des-Arg9-BK levels in the serum of patients with MMD.
Next, we determined the effect of BK, B1R and des-Arg9-BK on angiogenesis and BBB integrity.Our results could shed light on MMD pathogenesis and the potential of BK as a potential target for treating patients with MMD.internal carotid and middle as well as anterior cerebral arteries along with an atypical collateral vessel network. 20,21The patients were excluded based on the following criteria: patients with secondary MMD due to atherosclerosis, hyperthyroidism, neurofibromatosis, meningitis, leptospiral infection or prior skull-base radiation therapy.Healthy controls (HCs) recruited from hospitals were age-and sex-matched.All experiments including human participants were performed in accordance with the Helsinki Declaration and were approved by the medical ethics committee of the Jining First People's Hospital.All participants or their guardians provided informed consent.

| Sample collection
We collected small fragments of superficial temporal artery (STA) tissue during the surgery.We collected blood from patients before and during the surgery using lithium heparin-containing tubes and centrifuged the blood samples.Next, we collected and stored the serum at −80°C for future analysis.We fixed vascular tissues in 4% paraformaldehyde (PFA) for 24 h, embedding them in paraffin and sectioning them for further examination.
We obtained and stored the serum of participants from the HC group following the same method.These experiments were performed following established protocols to ensure the accuracy and reliability of the collected samples.
Briefly, we added serum samples and standards to antihuman monoclonal antibodies-coated wells, sealed the plates with a membrane and incubated them at 37°C for 30 min.Next, we added a secondary horseradish peroxidase (HRP)-labelled antibody to all wells and incubated them for an antigen-antibody-HRP complex formation.Furthermore, we washed the wells, added the substrate solutions A and B, and incubated them for 10 min.The reaction was then stopped, resulting in a yellow colour.All analyses were performed in duplicates, and the results were averaged.
DMEM contained 4500 mg/L D-glucose, 110 mg/L sodium pyruvate, L-glutamine and 1.5 g/L sodium bicarbonate.Once cells were confluent, they were treated with 100 nM BK or des-Arg-BK for 2 h.

| Immunofluorescence (IF)
For antigen retrieval, we deparaffinized the tissue sections with xylene and hydrated them with descending gradients of ethanol.We fixed and permeabilized bEND.3 cells with ice-cold methanol and acetone, respectively.

| Evaluation of blood-brain barrier in vitro model-transendothelial electrical resistance (TEER) and transcytosis assays
We seeded the cells on 0.4 μm pore size inserts in a 24-well transwell plate (Corning) for 1 week to establish a blood-brain barrier model in vitro, 22 and we evaluated its integrity by the measurement of TEER values daily as previously described. 23The total resistance (Ω) was measured using an STX2 probe of the EVOM2 system.We normalized all TEER values to the area of the membrane, and the values were corrected for the resistance without cells.We performed all experiments in duplicates and a minimum of three independent differentiations.We plotted TEER versus time graphs for normalizing TEER values to the peak value of the control, such that the maximum relative TEER value of each biological replicate of the control reached 1.0 at its highest value.A TEER value greater than 200 Ω*cm 2 indicated the successful establishment of the model, allowing for subsequent drug administration experiments.
The permeability of BBB model was assessed once the cell monolayer had formed a dense layer.In the upper chamber of the transwell insert, we added 0.1 mL of a solution containing 0.5 mg/ mL FITC-dextran (prepared in serum-free, phenol red-free DMEM), while in the lower chamber, we added 0.6 mL of a solution containing 0.5 mg/mL non-FITC-labelled dextran (also prepared in serum-free, phenol red-free DMEM).After incubating in the cell culture incubator for 1 h, we separately extracted samples from the upper and lower chambers of the transwell device for fluorescence intensity measurements.The permeability coefficient (Pd) of the cell layer was calculated using the following formula: Pd (cm/s) = (C/t) × (1/A) × (V/L).
Where: C represents the concentration of FITC-dextran in the lower chamber, t is the time interval, A is the surface area, V is the volume of the lower chamber solution and L is the concentration of FITC-dextran in the upper chamber.

| Angiogenesis assay
In conditioned media, endothelial cells respond to angiogenic signals by forming capillary-like structures.The process of tube formation occurs rapidly, with endothelial cells aligning themselves and lumencontaining tubules beginning to appear within a remarkably short timeframe.The tube formation assay was proposed as a rapid, reproducible and highly sensitive in vitro method for measuring angiogenesis. 24First, cells were treated, and bEnd.3 cells were seeded at a density of 1 × 10 5 cells/well in a 24-well plate coated with Matrigel and incubated for 8 h under normal conditions.Next, we selected and imaged five representative fields.For quantitation, total tube length and number of tube branches were calculated using ImageJ software.All experiments were performed in triplicate and analysed blindly.

| Migration assay
For the migration assay, we seeded bEnd.3 cells on the upper chamber of a transwell membrane in 24-well plates for 24 h.Next, cells migrated to the lower chamber were fixed, stained and quantified with the aid of a microscope.All experiments were performed in triplicate.

| EdU test
We performed the EdU test to determine the effect of the BK and des-Arg9-BK on the proliferation of bEnd.3 cells.Briefly, 1 × 10 5 cells were cultured in DMEM+10% FBS for 24 h.First, we incubated the cells with EdU-labeling reagent (1:1000, Invitrogen) for 2 h, discarded the medium, and washed the cells with phosphate-buffered saline (PBS) twice for 5 min.Next, we fixed the cells with 4% PFA and incubated them with 2 mg/mL of glycine for 5 min, respectively.The cells were rinsed with PBS for 5 min and stained with the Click-iT Edu Alexa Fluor 555 Imaging Kit (Invitrogen) and DAPI (1:500, Invitrogen) for 5 min.Finally, we examined the nuclei using a fluorescence microscope and determined the count of EdU+ cells.

| Statistical analysis
We statistically analysed the data using the Statistical Package for Social Sciences (SPSS) version 19.0 software (SPSS Inc., Chicago, IL, USA).Next, an independent t-test was employed to compare the baseline levels of the two groups for normally distributed variables.Variables were compared using a t-test or Mann-Whitney U test as appropriate to determine the presence of MMD, and multiple logistic regression analysis (MLRA) was conducted.We performed receiver operating characteristic (ROC) curve analysis to split continuous variables using a calculated cut-off value.All histograms were plotted using GraphPad Prism 9.0.p < 0.05 was considered statistically significant.

| Demographics of subjects
We enrolled 51 patients with MMD (26 males and 25 females) and 65 matched HCs (39 males and 26 females).MMD and HC (p > 0.05).The situation for the type of onset and the pathogenic site were summarized for patients in the MMD group.
MLRA was conducted to determine if BK, des-Arg9-BK and B1R could predict MMD risk.We dichotomized the continuous variables based on a cut-off value calculated using the ROC curve.The area under the ROC curve value of BK was 0.665 (p = 0.001), des-Arg9-BK was 0.752 (p < 0.001) and B1R was 0.642 (p = 0.006, Figure 1D,F).

| Increase in B1R expression in the STA tissues of patients with MMD
We measured B1R expression by IF (Figure 2).The results revealed an increase in B1R expression in STA tissues of patients with MMD compared to HCs, consistent with quantitative analysis in the serum of the participants.B1R intensity in the STA tissues of patients with MMD was approximately 1.6 times higher compared to HCs.

| BK and des-Arg9-BK Limit bEnd.3 Cell angiogenesis
BEnd. blood vessels, recapitulates several angiogenesis features in vivo. 25nce, we cultured bEnd.3 cells with BK and des-Arg9-BK individually to observe the effects of BK and des-Arg9-BK on cell angiogenesis.Figure 3A shows cell angiogenesis under different culture conditions.
Next, we quantified the angiogenesis parameters, including the rate of formation, branching and length of tubes (Figure 3B-D).The angiogenesis parameters of cells cultured with BK and des-Arg9-BK were significantly lower compared to the control group, indicating that BK and des-Arg9-BK could limit the formation of bEnd.3 cell tubes.These results suggest that bEnd.3 cells treated with des-Arg9-BK and BK could significantly inhibit angiogenesis.

| BK and des-Arg9-BK enhance the migratory and proliferative capacities of bEnd.3 cells
We determined the effects of BK and des-Arg9-BK on the migratory and proliferative capacities of bEnd.3 cells (Figure 4).BK causes the vasodilatation of arteries and arterioles via the endothelium in the brain and increases the permeability of the cerebrovascular system, thus causing vasogenic brain edema. 26,27s-Arg9-BK is an active BK metabolite that affects single pial venular capillaries.B1R and B2R are BK receptors activated by BK and des-Arg9-BK, respectively.Several studies have determined the correlation between BK, BK receptors and cerebrovascular occlusive diseases, such as cerebral ischemia, stroke and cerebral infarction. 26,28,29To the best of our knowledge, no studies have demonstrated a correlation between BK, BK receptors or des-Arg9-BK and MMD pathogenesis.Our results revealed a significant increase in BK, B1K and des-Arg9-BK levels in the serum of patients with MMD compared to HCs, thus suggesting BK, B1K and des-Arg9-BK could be involved in MMD pathogenesis.Next, IF results showed a significant increase in B1R expression levels in the STA tissues of patients with MMD, thus indicating the involvement of B1R in MMD pathogenesis.Previous studies have shown that BK and B1R could promote the release of proinflammatory cytokines in the microvascular endothelial cells of the brain 18,30 and B1R activation induced inflammation in human brain microvascular endothelial cells. 29We have previously demonstrated a significant increase in interleukin-1β, tumour necrosis factor and interleukin-12 levels in the serum of patients with MMD compared to HCs. 31 Therefore, the present study is an extension of our previous study to explore additional mechanisms involved in MMD pathogenesis.
Studies have used immunohistochemistry to detect CD31 expression for demonstrating the presence of endothelial cell tissue and evaluating angiogenesis. 32Our IF results showed an increase in B1R expression levels in the STA tissues of patients with MMD.
These results indicate that B1R and their upstream BK and BK metabolites could affect MMD pathogenesis.Therefore, we established an in vitro model to verify our hypothesis.
BK and des-Arg9-BK demonstrated significant inhibitory effects on the formation, branching and length of the vessel in vitro, thus indicating a significant inhibitory effect of BK and des-Arg9-BK on angiogenesis.These results strongly support our initial hypothesis.
Previous studies have reported that BK and des-Arg9-BK regulate angiogenesis.However, there are discrepancies between previous studies and our results.Wang et al. showed that BK promotes angiogenesis. 33Additionally, another study demonstrated that BK increases vascular permeability during the early phase via B2R in the endothelial cells to promote angiogenesis. 34Moreover, BK promotes angiogenesis and the growth of tumours in vivo. 35

| CON CLUS ION
In this study, we demonstrated an increase in BK, des-Arg9-BK and We enrolled patients with MMD receiving treatment at the Department of Neurosurgery of the Jining First People's Hospital.Patients with MMD were diagnosed based on strict diagnostic criteria outlined by the Research Committee on Spontaneous Occlusion of the Circle of Willis of the Ministry of Health, Labor, and Welfare in Japan.The diagnostic criteria included severe occlusion or stenosis of the and B1R levels in the serum and the brain could be potential strategies for treating patients with MMD.K E Y W O R D S blood-brain barrier, bradykinin, bradykinin receptor, des-Arg9-BK, Moyamoya disease | 4071 WANG et al.
3 cells cultured on 3D ECM form capillary-like structures, or tubes, thus indicating the ability of cells to form the inner lining of F I G U R E 1 Levels of Bradykinin (BK) (A), Des-Arg9-BK (B) and B1R (C) in the serum of patients with MMD and healthy subjects.Data are expressed as the mean ± SD. **p < 0.01.ROC curve analyses of the Bradykinin (BK) (D), Des-Arg9-BK (E) and B1R (F) ROC, receiver operating characteristic; AUC, area under the curve.

F I G U R E 3
Bradykinin (BK) and Des-Arg9-BK Limit Tube Formation bEnd.3 cells.Representative images (A-C), Scale: 100 μm.Tube formation rate (D), relative branch points (E) and relative total tube length cultured with BK and des-Arg9-BK compared with controls **p < 0.01.
Guo et al. demonstrated the molecular mechanisms mediating the proangiogenic activity of BK.The use of different cell types in these studies could be the underlying cause of discrepancies in results.36Hence, further studies are required to determine the effect of BK on angiogenesis.Moreover, our results showed that BK could enhance the migratory and proliferative capacities of cells, consistent with previous studies using different cells.Studies have reported that BK stimulates or promotes the migration and proliferation of gastric cancer, human cardiac progenitor and cervical cancer cells.Further, BK regulated the migratory and proliferative capacities of cells via the ERK and STAT3 signalling pathways.37,38These results shed light on the significance of BK in MMD pathogenesis.BBB plays a crucial role in the maintenance of homeostasis in the brain.Numerous studies have investigated the correlation between BBB disruption and MMD pathogenesis.A study used sodium fluorescein videoangiography to demonstrate BBB impairment in patients with MMD.17Moreover, Lu et al. showed a significant decrease in cortical perfusion in patients with MMD and disrupted BBB compared to patients with intact BBB.Furthermore, significant disruption of the BBB was observed in patients with MMD compared to patients with arteriosclerotic cerebrovascular disease. 39Studies have investigated the application of several biomarkers, including VEGF, MMPs, claudin 5, occludin and junction-associated molecule-1, for assessing BBB function in patients with MMD.In this study, we investigated the effects of BK and des-Arg9-BK on BBB integrity and the regulation of TJ proteins to determine the role of BK and des-Arg9-BK in the pathogenesis of MMD.Our results revealed that BK and des-Arg9-BK could disrupt BBB integrity and decreases TJ protein expression levels.Together, these findings indicate that BK and des-Arg9-BK could be involved in MMD development by impairing BBB integrity.However, the present study has a few limitations.First, our sample size was relatively small, and the cross-sectional study failed to determine a causal correlation.Hence, additional large sample-size longitudinal studies are required to validate our results.Second, efficient MMD animal models are lacking.Hence, we performed in vitro studies to determine the mechanism underlying the effect of BK and des-Arg9-BK on MMD.Additional studies using animal models or human specimens are required to determine the mechanism of BK and des-Arg9-BK in patients with MMD.Finally, the correlation between MMD pathogenesis, the proliferation and migration of cells, angiogenesis and BBB impairment should be further explored.These efforts would provide additional evidence to support our findings.

B1R levels in theF I G U R E 4
serum and the STA tissues of patients with MMD, thus indicating that these factors could contribute to MMD pathogenesis.Our results showed that BK and des-Arg9-BK could inhibit angiogenesis, promote the proliferation and migration of cells and disrupt BBB integrity.Thus, regulating the BK, des-Arg9-BK and B1R levels in the serum and the brain could be potential strategies for treating patients with MMD.Bradykinin (BK) and Des-Arg9-BK promote bEnd.3 cells migration and proliferation.(A,B) Transwell assay was performed to evaluate cell migration, Scale: 100 μm; the number of migrated cells was recorded compared with controls.(n = 3) (C,D) Cell proliferation was measured via EdU staining, Scale: 20 μm; Quantification of Edu/DAPI compared with controls based on the immunofluorescent images.(n = 3).**p < 0.01 ns, not significant.F I G U R E 5 Bradykinin (BK) and Des-Arg9-BK distrupt BBB barrier permeability in vitro.s.(A) Pictorial representation of TEER and FITCdextran translocation assays.(B) Relative TEER values in different culture through the time postseeding.(C) FITC-dextran permeability across the bEnd.3cells.(D) Immunofluorescence staining for Claudin-5 and ZO-1 in cultured Bradykinin (BK), Des-Arg9-BK and control.(E) Representative Fluorescence intensity of Claudin-5 and ZO-1 in cultured Bradykinin (BK), Des-Arg9-BK and control.(F) Representative Western blot of Occludin, Claudin-5 and ZO-1 in cultured Bradykinin (BK), Des-Arg9-BK and control.(G) Quantification of Occludin, Claudin-5 and ZO-1 in cultured Bradykinin (BK), Des-Arg9-BK and control, respectively.

Table 1
Demographic and clinical characteristics of the study subjects.
shows detailed demographic characteristics, including gender, age (in years), body mass index (BMI) and total number of participants.The results showed no significant difference in age, sex and BMI of patients with TA B L E 1