Delayed changes in the transcriptomic profile of cerebral arteries in a rat model of subarachnoid hemorrhage

Experimental Neurology Delayed changes in thetranscriptomic profileof cerebral arteries in a rat model of subarachnoid hemorrhage --Manuscript Draft-- Manuscript Number: EXNR-24-735R1 Article Type: Research Paper Section/Category: Cellular and Molecular Neuroscience Keywords: Subarachnoid Hemorrhage (SAH); cerebral arteries; microarray analysis; functional enrichment; immune response Corresponding Author: Antonio Castellano, Ph.D. University of Seville SPAIN First Author: Gonzalo Revilla-González Order of Authors: Gonzalo Revilla-González María del Carmen González-Montelongo Elton JR Vasconcelos Juan Ureña Jian Shi Antonio Castellano, Ph.D. Abstract: Aneurismal subarachnoid hemorrhage (aSAH) is a neurovascular disease characterized by blood released into the subarachnoid space due to rupture of the cerebral arteries. After the onset of bleeding, secondary associated vasospasm (VSP) remains a dramatic side effect that causes severe comorbidities. We analyzed alterations in the expression profiles of arteries from a rat model of SAH using microarray and bioinformatics approaches. A single injection autologous blood rat model, previously characterized in our laboratory, was used. We performed a total RNA extraction and a microarray analysis of cerebral arteries from animals 7 days after surgery to study the delayed transcriptional changes induced by SAH. To assess the functional relationship between differently expressed genes, we run a combination of gene enrichment tools: GSEA, ClueGO, and ClusterProfiler. Our results showed that in SAH animals, the gene sets related to inflammation and immune system activation were up-regulated; genes related to the pathways involved in the regulation of muscle contraction had their expression disturbed; and the gene categories associated with DNA damage and repair were overrepresented. In conclusion, our results suggest that, after the SAH insult, multiple mechanisms, rather than a single cause, are activated at the same time in the cerebral vessels to trigger vascular alterations. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation SAH Animal model Single injection of autologous blood in Cisterna Magna model. Cerebral arteries isolation and RNA extraction was done 7 days after surgery (long-term). Our results suggest that, long-term after SAH insult, vascular alterations and VSP could be triggered by disrupting multiple mechanisms at the same time in the cerebral vessels. Our data provide new clues about the mechanisms underlying the changes induced by SAH and will help pave the way for new basic and applied research on this pathology. Transcriptional and enrichment analysis of arteries from the SAH animal model Inflammation and immune system alterations were up-regulated and enriched longterm after bleeding. Pathways related to vasospasm (VSP) were disturbed and DNA damage-producing processes induced. Graphical Abstract Delayed changes in the transcriptomic profile alterations of cerebral arteries in a rat model of subarachnoid hemorrhage Gonzalo Revilla-Gonzáleza,b,c,+,*, María del Carmen González-Montelongoa,b,e,f,†,*,#, Elton JR Vasconcelosd, Juan Ureñaa,b, Jian Shic,&, Antonio Castellanoa,b,#,& aInstituto de Biomedicina de Sevilla, IBIS/ Hospital Universitario Virgen del Rocío/CSIC/ Universidad de Sevilla. Sevilla, Spain. bDpto. Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, Spain. cLeeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), School of Medicine, University of Leeds. Leeds LS2 9JT. United Kingdom. dLeeds Omics, University of Leeds. Leeds LS2 9JT. United Kingdom. eInstituto de Investigación e Innovación Biomédica de Cádiz (INiBICA), Unidad de Investigación, Hospital Universitario Puerta del Mar, Av. Ana de Viya 21, 11009 Cádiz, Spain. fÁrea de Fisiología, Facultad de Medicina, Universidad de Cádiz, Spain. *These authors share the first authorship. &These authors share the last authorship. Gonzalo Revilla-González: g.revilla[email protected].uk. ORCID: 0000-0002-5512-8381 +Present address: Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM), School of Medicine, University of Leeds. Leeds LS2 9JT. United Kingdom. Jian Shi: [email protected]. ORCID: 0000-0003-2179-8579 Elton J. R. Vasconcelos: [email protected]. ORCID: 0000-0001-5130-6622 Juan Ureña: [email protected] ORCID: 0000-0003-4086-5941 María del Carmen González-Montelongo: [email protected] / mariadelcarmen.go[email protected]. ORCID: 0000-0003-2332-6153 †Present address: Unidad de Investigación, Instituto de Investigación e Innovación Biomédica de Cádiz (INiBICA), Hospital Universitario Puerta del Mar, Avda. Ana de Viya 21, 11009 Cádiz, Spain; and Área de Fisiología, Facultad de Medicina, Universidad de Cádiz, C/Dr. Marañon 3, 3rd Floor, 11002, Cádiz, Spain. Antonio Castellano: [email protected]. ORCID: 0000-0003-3955-5137 #Correspondence to: ac[email protected], Tel.: +(34)-955923059 (A.C.); [email protected], Tel.: +(34)-956003111 (M.C.G.M.) Highlights  SAH induces delayed changes in gene expression in the cerebral arteries.  Inflammation and the immune system are up-regulated on day 7 after SAH.  The pathways related to vasospasm (VSP) are altered after bleeding.  Vascular alterations and VSP could be produced by multiple mechanisms after SAH. Abstract Aneurismal subarachnoid hemorrhage (aSAH) is a neurovascular disease characterized by blood released into the subarachnoid space due to rupture of the cerebral arteries. After REVISED Manuscript Click here to view linked References the onset of bleeding, secondary associated vasospasm (VSP) remains a dramatic side effect that causes severe comorbidities. We analyzed alterations in the expression profiles of arteries from a rat model of SAH using microarray and bioinformatics approaches. A single injection autologous blood rat model, previously characterized in our laboratory, was used. We performed a total RNA extraction and a microarray analysis of cerebral arteries from animals 7 days after surgery to study the delayed transcriptional changes induced by SAH. To assess the functional relationship between differently expressed genes, we run a combination of gene enrichment tools: GSEA, ClueGO, and ClusterProfiler. Our results showed that in SAH animals, the gene sets related to inflammation and immune system activation were up-regulated; genes related to the pathways involved in the regulation of muscle contraction had their expression disturbed; and the gene categories associated with DNA damage and repair were overrepresented. In conclusion, our results suggest that, after the SAH insult, multiple mechanisms, rather than a single cause, are activated at the same time in the cerebral vessels to trigger vascular alterations. Keywords Subarachnoid hemorrhage (SAH), cerebral arteries, microarray analysis, functional enrichment, immune response. Introduction Aneurismal subarachnoid hemorrhage (aSAH) is a neuropathology that occurs when the brain arteries rupture and subsequently blood is released into the subarachnoid space. Although its worldwide incidence has decreased from 10.2 per 100,000 person-year in 1980 to 6.1 in 2010, it is still a major neurovascular disease with a high case fatality rate (Etminan et al., 2019). Patients who survive the hemorrhagic insult have a high diseasespecific burden; up to 19 % will become disabled for work and could suffer cognitive, functional and behavioral complications (Al-Khindi et al., 2010; Eagles et al., 2019; Nieuwkamp et al., 2009). Damages after aSAH are classified as early cerebral injury (ECI), those that occur up to 72 h after the insult, or delayed cerebral injury (DCI), those appearing from day 3 up to 14 after hemorrhage (Macdonald and Schweizer, 2017). DCI is produced by artery occlusion and cerebral damage in poorly irrigated regions of the brain. One of its main causes is cerebral vasospasm (VSP), which occurs as a consequence of the effect of the released blood on the walls of the vessels (Brami et al., 2019; Crowley et al., 2011; Egea-Guerrero et al., 2015; Mossa-Basha et al., 2019). Cerebral arteries are structured in three main layers, whose alterations could induce VSP under pathological conditions. VSP could be produced by increasing myocyte contractility via Ca2+-dependent and Ca2+-independent mechanisms. However, in some vascular pathologies it can also be produced by activating molecular pathways related to inflammation and vascular remodeling (Liu and Khalil, 2018). Existing studies on cerebral artery transcription profiles after SAH are limited. In rat models of ECI, an increase in the expression of genes related to inflammation, extracellular matrix remodeling, and apoptosis was observed (Vikman et al., 2007, 2006). Furthermore, in a canine model of DCI, genes related to inflammation and Ca2+ regulation were also up-regulated (Sasahara et al., 2008). The purpose of the present work was to investigate, using transcriptomic assays and bioinformatic approaches, SAH-induced delayed alterations in the gene expression profile of the rat cerebral arteries that could explain the pathological processes that cause VSP. Material and methods Animals Male wistar rats (400 g approximately and six months old) were divided into three groups: control, sham7d, and SAH7d. We have analyzed this time point according to previous results from our laboratory showing an increase in smooth muscle myocyte excitability on day 7 after surgery (Revilla-González et al., 2024a). The experiments were carried out according to the Spanish legislation on the protection of experimental animals and were approved by the local animal care committee according to the European Directive 2010/63/EU and the Spanish Royal Decree 53/2013. Before and after surgery, the animals were housed in separate cages in a dry place, away from sources of infection and surgical areas. A stable temperature between 23 and 27 ºC and a light/dark cycle were maintained for 12 h. They received food and water ad libitum before and after surgery. Surgical procedures and sacrifice Surgery was performed following procedures previously published by our group (EgeaGuerrero et al., 2015; Muñoz-Sánchez et al., 2012). Before procedures, the animals were anesthetized with a preparation composed of 96 µg/g Ketamine (Ketolar©, Pfizer, 50 mg/mL), 4.5 µg Xylazine (Rompun©, Bayer, 20 mg/mL) and 0.2 µg/g Atropine (Atropine Bayer© 1 mg/mL). The optimal dose to achieve a good degree of analgesia and sedation (2.5 µL/g) was injected intraperitoneally. Subsequently, the head, neck, and tail were shaved and the animals were immobilized in a decubitus-prone position in a stereotaxic frame (Stoelting©). During surgery, animal breathing, temperature, and an appropriate degree of sedation and analgesia were continuously monitored. Sham and SAH groups were generated using a 25G needle (BD Biosciences) and 1 mL syringe (BD Plastipak©), mounted on the arm of the stereotaxic frame. 100 µL of cerebrospinal fluid was removed from the cisterna magna through the posterior atlanto-occipital membrane. Then, in the SAH group, 100 µL of blood was obtained from the caudal vein and immediately injected intracisternally. In the sham group, the same procedures were performed, but instead of blood, the same volume of saline (0.9 % NaCl) was injected. After surgery, both animals were returned to their cages to monitor the proper awakening. The control group did not receive any surgery. On day 7 the animals were sacrificed by exsanguination after injection of a lethal dose of sodium thiopental. For RNA analysis, we followed the extraction method previously reported (Revilla-González et al., 2024a). Large arteries of the cerebral surface directly exposed to blood were used. Whole brains were extracted and the arteries were cleaned under a magnifying glass in ice-cold Hank’s solution free of RNAses, immediately frozen in liquid nitrogen and stored at -80 ºC. Hank’s solution composition was (in mM): 125 NaCl, 5.36 KCl, 0.44 KH2PO4, 0.34 Na2HPO4, 5 NaHCO3, 10 glucose, 1.45 sucrose, 10 HEPES, at pH 7.4. Total RNA extraction and microarray analysis The homogenization of the arteries was performed using a Polytron PT4000 (Kinematica) for 5 seconds in RLT buffer from the RNeasy® Micro Kit (Qiagen). Subsequently, the samples were incubated at room temperature for 5 min and then centrifugated at 12,000 X g, for 3 min, until the supernatant was clarified. The RNA isolation process was completed using the QIAcube platform (Qiagen). Once the RNA was isolated, it was eluted in 15 µL of RNAses-free water, quantified using a Nanodrop 2000 (Thermo) and finally frozen at -80 ºC. Total RNA was amplified and labeled using the GeneChip® WT PLUS Reagent Kit (Thermo). Amplification was carried out using 100 ng of total RNA. The amplified cDNA was quantified, fragmented and labeled for hybridization with GeneChip® Clariom S Rat Array (Thermo). The arrays were washed and labeled in a GeneChip® Fluidics Station 450 (Thermo) and scanned with GeneChip® Scanner 3000 (Thermo). Finally, data were analyzed with the TAC v4 program (Thermo), setting the P-value < 0.05 and Fold-change > 1.3 as thresholds for Differentially Expressed Genes selection (DEGs). Differences between groups were evaluated using eBayes test. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE266601. Validation of microarray data using RT-qPCR Data from the microarray analysis were validated by RT-qPCR. A detailed description of the validation process is included in “Supplementary materials”. Enrichment analysis DEGs lists were submitted to a gene set enrichment assay (GSEA) with GSEA v4.1 (Mootha et al., 2003; Subramanian et al., 2005) and the following libraries: KEGG C2 v7.4, Reactome C2 v7.4, GO Biological processes C5 v7.4 y GO Molecular Functions C5 v7.4. Additionally, overrepresentation analyzes were performed using the ClusterProfiler Bioconductor package (Wu et al., 2021) and ClueGo (Bindea et al., 2009) as alternative tools for more comprehensive functional analysis elucidation. As in the GSEA analysis, GO, KEGG, and Reactome were the main functional annotation consortia relied on, setting an adjusted P-value threshold of 0.05 for both tools. Results Sample distribution in a SAH animal model To assess changes in gene expression caused by SAH, we have performed a microarray analysis to characterize gene expression profiles in the three sample groups under study: control, sham7d, and SAH7d. Principal component analysis (PCA) showed clear differences between the different groups. The control and SAH7d groups were placed in distal positions, indicating important differences in their expression profiles, while the sham7d group was located halfway, indicating an intermediate expression profile (Fig. 1A). The following control tests were run to verify the quality of the samples: positive vs negative AUC, to compare the intron controls to the exon controls; hybridization control, to evaluate sample hybridization efficiency on gene expression arrays; and labeling controls, to monitor the target labeling process. All samples used in the study passed these screenings, allowing their use for further analysis (Fig. 1B-D). Fig. 1 PCA analysis and quality control. (A) PCA analysis showing the distribution of the samples for each group. The control and SAH7d groups were placed in distal positions, while the sham7d group occupied an intermediate position. (B-D) Pos vs Neg AUC, hybridization and labeling control tests were used to analyze the quality of the different samples. Every sample passed quality control tests. Transcriptional changes in cerebral arteries after induction of SAH Once we confirmed the distribution of the samples and the quality of the microarrays, we performed a gene differential expression analysis using TAC4 software to study how the gene expression profile could be altered after SAH. For this purpose, we considered as differentially expressed genes those with a fold-change <-1.3 and >1.3 and a P-value <0.05. Volcano plots show the distribution of the differentially expressed genes (Fig. 2A). A total number of 23,188 genes were analyzed. Fig. 2B shows the number of genes differentially expressed in a two-by-two comparison of the experimental groups, and details of the gene distribution are summarized in Table 1. In the comparison of SAH7d vs. control, a total number of 1,706 genes were differentially expressed, 801 were up- regulated and 905 were down-regulated under the SAH condition. In sham7d vs. control, 1,246 genes were differentially expressed, 522 were up-regulated, and 724 were downregulated in sham. Finally, in SAH7d vs. sham7d, 817 genes were differentially expressed, where 438 genes were up-regulated and 379 were down-regulated in SAH group. The Venn diagram shows the logical relationships between the differentially expressed genes for each comparison (Fig. 2C). Genes included in the AB area (226 genes) represent the group of interest. These genes were differentially expressed in both comparisons, SAH7d vs. control and SAH7d vs. sham7d, and were therefore genes whose expression changes are associated with bleeding (Supplementary Table 1). Among these genes, we have identified some that could be involved in various processes such as the functioning of Rho family GTPases and the activation and regulation of the immune system (details are summarized in Table 2). From the microarray data, we validated by RT-qPCR those genes included in the “Rho GTPases family and its regulation” and “activation and regulation of immune system” gene sets that showed the greatest change in expression (>1.5 fold-change) in the comparison of the SAH7d vs. sham7d groups. The RT-qPCR results confirmed, in most cases, the microarray data (Supplementary Figure 1). C3, IL10RA and LCP2, included in the “activation and regulation of immune system” gen set, that were up-regulated in the microarray analysis, were also significantly upregulated in the RT-qPCR validation. ARHGAP4, included in the “Rho GTPases family and its regulation” gen set and that was up-regulated in the microarray, showed a similar trend in the validation, although it did not reach statistical significance (P=0.103). However, the RAC2 gene, belonging to the same gene set, stood out, as it was the one that showed the greatest change in expression in the microarray, but did not show any significant change in expression in the qPCR validation. Fig. 2 Microarray analysis of genes that are up-regulated and down-regulated in the different comparisons. (A) Volcano plots show differences in expression between experimental groups. Fold-change <-1.3 and >1.3 and P-value< 0.05. (B) Differentially expressed genes between comparisons. Gray bars represent the total number of differential expressed genes (bars: red, up-regulated genes; green, down-regulated genes). The numbers above the bars indicate differentially expressed genes. (C) Venn diagram of the different comparisons showing the number of specific differentially expressed genes in each comparison. n= 3 control animals, n= 5 sham7d animals, and n= 5 SAH7d animals. SAH7d vs. control Total Passed Filter Up-Regulated Down-Regulated Coding 21848 1598 752 846 Multiple Complex 858 61 35 26 Non-Coding 310 25 3 22 Unassigned 142 17 7 10 Pseudogene 28 5 4 1 Precursor microRNA 2 0 0 0 sham7d vs. control Total Passed Filter Up-Regulated Down-Regulated Coding 21848 1186 495 691 Multiple Complex 858 33 20 13 Non-Coding 310 17 2 15 The activation of the immune system and inflammation after bleeding are side effects observed in both patients and animal models of SAH (Gris et al., 2019; Ridwan et al., 2021; Savarraj et al., 2018). Furthermore, activation of immune processes in cerebral vessels is considered one of the causes of SAH (Li et al., 2017; Wang et al., 2017). Our microarray results, validated using RT-qPCR, showed that inflammatory pathways were altered in SAH7d animals vs. control and sham7d groups, and were consistent with previous microarray studies performed in earlyand long-term SAH animal models (Sasahara et al., 2008; Vikman et al., 2007, 2006). In addition, our results align with bioinformatic studies performed in human brain and aneurismal wall tissues showing the activation of immune system in the subarachnoid hemorrhage environment (Kurki et al., 2011; Rosell et al., 2011; Ye et al., 2022). An increase in long-term immune response after aSAH is also observed in patients (Bacigaluppi et al., 2020), and recent results from our laboratory showed an increase in the number of monocytes and their adhesion to the endothelium, 5 days after bleeding in patients who developed VSP (Revilla-González et al., 2024b). The enrichment of these categories, together with these previous results, is a relevant finding in this pathology. The activation of the immune response and the inflammatory processes induced by SAH in the cerebral arteries could be related to VSP and a poor prognosis of patients after SAH. These processes could be targeted in new therapeutic approaches that allow better patient management. The Rho family is a family of small GTPases that mediates several intracellular signaling processes, including VSMC contraction or relaxation through the Ca2+ sensitization mechanism (Liu and Khalil, 2018). Our microarray results showed an increase in the expression of genes associated with negative regulation of these proteins (ARAP3 and ARHGAP4) and a decrease in genes associated with positive regulation (ARHGEF4). Furthermore, other genes that codify other members of this family were up-regulated (RAC2 and RHOG). RT-qPCR validation data showed a positive trend in ARHGAP4 that matched the microarray results. However, RAC2 expression levels in SAH animals did not show this correlation. The reasons that could explain this discrepancy may include differences in the sensibility and specificity of the techniques, background or false positives in microarray, the low number of samples and differences in the sample quality. Previous results from our laboratory showed that the RhoA/ROCK pathway is functionally involved in the regulation of the smooth muscle contraction in response to long depolarizations and in vasoconstriction of the cerebral arteries of SAH animals 5 days after bleeding (Egea-Guerrero et al., 2015). Furthermore, the results of experiments carried out in our laboratory on peripheral blood mononuclear cells from patients showed that RhoA function is significantly increased 4 days after bleeding in patients who developed VSP (González-Montelongo et al., 2018). These results suggest that there is a time window for RhoA/ROCK activation, peaking 4-5 days after SAH and then declining. These observations are consistent with a previous work that described a peak in RhoA expression on day 5 and a slight decrease on day 7 after SAH (Miyagi et al., 2000). Other authors have pointed out an increase in RhoA translocation to the plasma membrane in acute cell cultures and animal models of SAH, and an enhancement effect mediated by endothelin-1 (Chang et al., 2014; Wang et al., 2014; Wickman et al., 2003). All this evidence suggests that the expression and function of the RhoA/Rock pathway have a dynamic behavior over time, acting in parallel with other pathways involved in SAH. Extravasation of blood from cerebral vessels in SAH produces deleterious effects in SAH. These effects can be assessed by measuring the expression of some proteins related to brain injury (Matz et al., 1996). Our results of the GSEA analysis performed in cerebral arteries showed that the gene sets related to DNA damage and repair were enriched in the SAH7d group relative to the control and sham7d groups, indicating that hemorrhage activates the processes related to cell injury and death in myocytes. This observation is in agreement with previous results that showed an increase in DNA fragmentation and cell injury in brains from earlyand delayed injury animal models of SAH (Matz et al., 2001, 2000, 1996). In addition, a previous microarray study on early effects performed in a rat model indicated that genes related to apoptosis were also up-regulated in pathological animals (Vikman et al., 2006). These results suggest that DNA damage and apoptosis are processes that occur after SAH. However, more research is needed in this field to determine if these processes are the cause or effects of pathophysiological alterations. In summary, our experiments show that gene expression is altered in the cerebral arteries after SAH. Processes related to inflammation and immune system activation are upregulated and enriched 7 days after bleeding. Gene categories related to pathways that can influence the VSP process are also disturbed, and processes that induce DNA damage in cells are induced. These results suggest that, after SAH insult, vascular alterations and VSP could be triggered by disrupting multiple mechanisms at the same time in the cerebral vessels, supporting the interaction between them instead of a single cause for this neurovascular disease. These results also support that our animal model of SAH, induced by autologous intracisternal injection of blood, is valid and reproduces many of the changes observed in patients with SAH and other animal models. Conclusions Classically, attempts have been made to identify a single cause of vasospasm after SAH, but this strategy has not resolved VSP in a high percentage of patients. However, our results support that a concert of alterations that include changes in the immune system, regulation of contraction, and cellular integrity occur at the same time after the hemorrhagic insult. This highlights that the etiology of vasospasm could be the combined effect of several alterations, which implies that its therapeutic approach should include multiple perspectives. Our results provide new clues about the mechanisms underlying the changes observed after SAH and will help to pave the way for new fundamental and applied research on this pathology. Funding The study was supported by the Research Grants SAF2017-89474-R Ministerio de Economía, Industria y Competitividad, Spain; JU and AC) and US-1381231 (Proyectos I+D+I FEDER Andalucia 2014-2020; AC). This study is partly supported by the British Heart Foundation Project Grant (PG/21/10595; J.S.). GR-G is the recipient of a predoctoral fellowship of the “V Plan Propio de la Universidad de Sevilla” and postdoctoral grant of the “Fundación Alfonso Martín Escudero”. Declaration of Competing Interest The authors declare no competing interests. Acknowledgement The authors acknowledge Dr. Francisco Jesús Morón-Civantos (Genomics Service, Instituto de Biomedicina de Sevilla (IBiS), Spain) for his technical support with the microarray; Raquel Gómez Díaz (Biomedical Research Support Service (SAIBIS) for her technical support in the RT-qPCR validation experiments; and Drs. Alberto Pascual and María Isabel Álvarez-Vergara (IBiS, Spain) for their advice on the GSEA technique. References Al-Khindi, T., Macdonald, R.L., Schweizer, T.A., 2010. Cognitive and Functional Outcome After Aneurysmal Subarachnoid Hemorrhage. 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(Cambridge 2, 100141. https://doi.org/10.1016/j.xinn.2021.100141 Ye, F., Liang, J., Wang, T., Wu, X., Li, J., Lan, K., Sheng, W., 2022. Bioinformatic Analysis of Coexpressed Differentially Expressed Genes and Potential Targets for Intracerebral and Subarachnoid Hemorrhage. World Neurosurg. 159, e442–e452. https://doi.org/10.1016/j.wneu.2021.12.070 Supplementary Material Click here to access/download Supplementary Material Article microarray SAH - Supplementary material R1.docx Declarations of interest: none Declaration of Interest Statement (Editable version)