Transcriptomic Profiling of the Hypothalamic PVN Reveals Sexually Dimorphic Pathways of Sympathoexcitation and Neuroinflammation in a Rat Model of Hypertension

doi:10.21203/rs.3.rs-7761756/v1

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Transcriptomic Profiling of the Hypothalamic PVN Reveals Sexually Dimorphic Pathways of Sympathoexcitation and Neuroinflammation in a Rat Model of Hypertension

https://doi.org/10.21203/rs.3.rs-7761756/v1

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Introduction: Hypertension is a multifactorial condition of unknown cause that affects more than 1.28 billion adults worldwide and impacts the sexes differently. The hypothalamic paraventricular nucleus (PVN) plays a central role in blood pressure regulation by modulating sympathetic tone and releasing neuropeptides that affect the function of the cardiovascular system. In this study, we investigated the transcriptomic profile of the PVN in hypertensive strains and across sexes, aiming to identify novel sex-specific molecular pathways involved in the regulation of blood pressure.

Methods: To accomplish this goal, we sequenced RNA from the PVNs of normotensive Wistar rats (WR) and Spontaneously Hypertensive Rats (SHR), both male and female. We also measured cardiovascular parameters and analyzed their variability.

Results: Cardiovascular analyses revealed greater blood pressure (BP) in SHRs than in Wistar rats andgreater participation of the sympathetic tone in SHR males than in their female counterparts. The impact of hypertension was also evident in the gene expression analyses, which revealed influences from both the hypertensive state and sex. Compared with female SHRs, male SHRs presented a marked increase in differentially expressed genes (DEGs), highlighting the greater impact of hypertension on the PVN transcriptome in males. Key upregulated genes in males, including Brain-Derived Neurotrophic Factor (Bdnf), Hypocretin (Hcrt), and Angiotensin II Receptor Type 1 (Agtr1a), are associated with elevated sympathetic tone. In contrast, the female transcriptomic signature was characterized by the upregulation of anti-inflammatory pathways, with significant upregulation of NLR Family CARD Domain Containing 3 (Nlrc3) and downregulation of Absent in Melanoma 2 (Aim2). Notably, several genes, such as Annexin A1 (Anxa1), whose downregulation is associated with neuroinflammation, were consistently downregulated in both sexes.

Conclusion: These results provide new insights into the cardiovascular and molecular basis of sex differences in hypertension, suggesting a greater contribution of sympathetic tone in males, whereas in females a greater anti-inflammatory component. These findings offer a valuable framework for developing future sex-specific therapeutic strategies.

Paraventricular Nucleus

Blood Pressure

Sex Differences

Transcriptomics

Inflammation

Anti-inflammatory

Sympathetic Tone

Cardiovascular

Hypertension

RNA-Seq

Hypertension is a complex condition of unknown cause that affects more than 1.28 billion adults worldwide and impacts the sexes differently. The hypothalamic paraventricular nucleus (PVN) plays a central role in blood pressure regulation by modulating sympathetic tone and releasing neuropeptides that affect blood pressure. In this study, we investigated the gene expression profile of the PVN in male and female Wistar rats and in Spontaneously Hypertensive Rats to identify the mechanisms involved in the regulation of blood pressure. RNA-sequencing analysis revealed that distinct gene expression profiles were influenced by sex and hypertensive conditions. We demonstrated that hypertensive male rats presented an increased number of differentially expressed genes, indicating a greater impact of hypertension on the PVN in males. The key upregulated genes in males included those linked to elevated sympathetic tone, consequently contributing to increased blood pressure. In contrast, the female transcriptome was characterized by the upregulation of anti-inflammatory pathways, which may mitigate brain inflammation in the PVN, contributing to increased blood pressure. Notably, genes associated with neuroinflammation were identified and found to be conserved between sexes. In conclusion, our data provide new insights into the key mechanisms underlying hypertension, as well as the sex effects of this condition. We demonstrated anti-inflammatory effects in females, which may help explain the lower prevalence of hypertension during the premenopausal period. These results offer a valuable framework for understanding the pathophysiology of hypertension and for developing future sex-specific therapeutic strategies.

Cardiovascular analyses confirmed elevated blood pressure in SHRs compared with Wistar rats, with male SHRs showing a stronger contribution of sympathetic tone than females.
Transcriptomic profiling of the PVN revealed a greater number of differentially expressed genes in male SHRs, underscoring the stronger impact of hypertension in males.
Consistent downregulation of Anxa1 across both sexes highlights a potential shared mechanism of neuroinflammation in hypertensive states.
Key upregulated genes in male SHRs, including Bdnf, Hcrt and Agtr1a, point to increased sympathetic activity as a central feature of the male transcriptomic profile.
Compared with male SHRs, female SHRs presented an anti-inflammatory signature characterized by Nlrc3 upregulation and Aim2 downregulation, which contrasts with the pro-sympathetic profile of male SHRs.

Primary hypertension, characterized by persistently high blood pressure (BP), is a global health crisis affecting over 1.28 billion adults aged 30–79 years worldwide¹. It is often known as a “silent killer” because of its asymptomatic nature and late diagnosis, leading to cardiovascular, cerebrovascular, and renal complications if left untreated^2–4. To prevent these complications, adequate and timely reduction of BP is recommended⁵. Current pharmacotherapies have limited efficacy⁶ and often require polypharmacy, especially in advanced stages of hypertension. Many genetic and environmental factors predispose individuals to the development of primary hypertension, but the fundamental causes remain elusive.

Central to the neuroendocrine and autonomic control of circulation is the paraventricular nucleus of the hypothalamus (PVN)^7,8. Its contribution to primary hypertension is well documented^9,10, operating through at least three pathways¹¹: magnocellular neurons (MCNs) of the PVN synthesize and secrete the neuropeptide hormone arginine vasopressin (AVP) into the circulation, where it increases BP by inducing vasoconstriction and regulating water balance, as confirmed in animal models^12–15 and humans¹⁶; the modulation of sympathetic outflow to the periphery via presympathetic parvocellular neurons (PCNs) that project directly to the intermediolateral nucleus (IML) to synapse preganglionic sympathetic neurons and indirectly via the rostral ventrolateral medulla (RVLM), increasing sympathetic outflow to the periphery^17,18; and the regulation of the stress response⁹ through corticotrophin-releasing hormone (CRH), which defines susceptibility to stress and predisposes individuals to hypertension^17,18. While under baseline physiological conditions, the PVN presympathetic PCNs are quiescent⁹, they become activated in hypertension through multiple mechanisms, including salt load; the mineralocorticoid receptor MR; angiotensin AT1R; V1aR; insulin; leptin; the downregulation of glutamate uptake by astrocytes; and the production of proinflammatory cytokines^10,13.

Significant sex differences exist in the prevalence and progression of hypertension, with premenopausal women generally exhibiting a protective phenotype¹⁹ compared with men²⁰. While the peripheral effects of sex hormones, particularly estrogen, are known to be protective¹⁹, the central neural mechanisms driving these differences are poorly understood. Sex hormone-dependent mechanisms have been discovered in the PVN that regulate AVP synthesis and release under basal conditions and stress. In the magnocellular part of the PVN, MCNs express the oestrogen receptor ERβ²¹, and oestrogen exposure has been found to lower the plasma osmotic threshold for AVP release²² as well as the threshold for the osmotic sensation of thirst²³. Indeed, estrogen and progesterone exposure are associated with plasma volume expansion and a leftward shift in the osmotic operating point for body fluid regulation^24,25. In the parvocellular part of the PVN, PCN also expresses ERα and ERβ and membrane-bound ER, which increases AVP and CRH synthesis^26,27. Numerous studies point to sex differences in the hypothalamic‒pituitary axis (HPA) and show that neurochemical reactivity to stress is greater in females than in males²⁸. Although many of the differences in BP regulation between sexes can be attributed to sex hormones, available clinical studies indicate that hormone replacement therapy in hypertensive postmenopausal women does not restore normal BP²⁹ suggesting a limited role of sex hormones and related mechanisms.

Given that the importance of the PVN as an integrative site for neuroendocrine and autonomic cardiovascular homeostasis is indisputable and that its contribution to the initiation and maintenance of hypertension is complex and multifactorial, we hypothesized that the PVN transcriptome may exhibit informative sexual dimorphism in the context of hypertension. We expected that spontaneously hypertensive (SHR) males would present a transcriptomic signature of enhanced sympathoexcitation and neuroinflammation, whereas SHR females would display a distinct, potentially protective, immunomodulatory profile. To test this hypothesis, we performed deep RNA sequencing of the PVN from hypertensive male and female rats and their normotensive counterparts, alongside a detailed cardiovascular autonomic assessment.

Animal Procedures

All experimental procedures in this study adhered to the guidelines set forth by Directive 2010/63/EU of the European Parliament concerning the protection of animals used for scientific purposes, as well as the UK Animals (Scientific Procedures) Act 1986. The experimental protocol was approved by the Ethics Review Board of the School of Medicine, University of Belgrade, and the relevant ministry of the Republic of Serbia (licence no. 119–01–13/17/2015–09). Furthermore, all studies involving animals were conducted in compliance with the ARRIVE guidelines for reporting animal experiments^30,31.

Male and female Wistar rats (WR) and SHR, aged 12 weeks (280–330 g), were housed in a controlled environment (12 h/12 h light‒dark cycle, temperature 21±2°C and humidity 65±9%) with free access to standard food pellets (0.2% w/v sodium content; Veterinarski Zavod, Subotica, Republic of Serbia) and tap water. The sample size (n = 6 per group) was determined via ‘power sample size calculation’ software (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize) to achieve a power of 90% and a type I error probability of 0.05 on the basis of expected effect sizes from prior studies.

Radiotelemetry Implantation and Hemodynamic Studies

The animals were anesthetized via a combination of ketamine (100 mg•kg−1, I.M.) and xylazine (10 mg•kg−1, I.M.). Rat body temperature was maintained by a heating pad (Harvard Apparatus, Holliston, MA, USA). Following medial abdominal 3 cm-long incision and intestine retraction, a catheter tip of the radiotelemetry device (TA11-PA C40; DSI, Transoma Medical, St Paul, MN, USA) was inserted into the aorta and fixed with 3 M VetbondTM and a tissue cellulose patch (DSI, Transoma Medical). Perioperatively, the rats were treated with gentamicin (25 mg•kg−1, i.m.) or carprofen (5 mg•kg−1 daily, s.c.) to reduce pain. Postoperatively, the animals were housed individually (30 × 30 × 30 cm Plexiglas cage) under controlled laboratory conditions. They were monitored every day until they fully recovered.

After a two-week recovery period, continuous cardiovascular hemodynamic measurements were recorded for one hour, a duration sufficient to capture a stable representation of autonomic tone, in four groups of animals: Wistar male rats (n=6), Wistar female rats (n=5, telemetric probe stopped working in one of the WR females), SHR males (n=6), and SHR females (n=6). Data acquisition from females was conducted during the dioestrus phase of the estrous cycle, as confirmed by daily examination of vaginal smears³². At the end of the registration period, the rats were euthanized by cervical dislocation and decapitated via a guillotine, and the brains were collected and stored at -80°C.

Cardiovascular signal processing and analysis

Arterial BP was digitalized at 1000 Hz in Dataquest A.R.T. 4.0 software (DSI, Transoma Medical). Systolic (SBP) and diastolic BP (DBP) and pulse interval (PI) or its inverse heart rate (HR) were derived from the arterial pulse pressure wave as the maximum, minimum and dP/dtmax interdistance, respectively. Spectral analysis of SBP, DBP and HR via fast Fourier transformation was performed after linear detrending, nine-point Hanning window filtering and resampling at 20 Hz³³. Spectra were obtained from 30 overlapping 2048-point time series of SBP, DBP and HR. The BP (mmHg2) and HR (bpm2) spectral powers were calculated for the whole spectrum (0.0195–3 Hz) and in three frequency ranges: very low frequency (VLF, 0.0195–0.195 Hz), low frequency (LF, 0.195–0.8 Hz) and high frequency (HF, 0.8–3 Hz). LF-BP and LF/HF-HR are established indicators of sympathetic outflow to arterial blood vessels and the sympathovagal balance of the heart, as observed in both experimental studies³⁴and clinical practice^35,36.

Evaluation of the spontaneous baroreceptor reflex via the sequence method

The method is explained in detail elsewhere³⁷. A stream of at least four consecutively increasing or decreasing SBP values and PI values delayed by three, four or five beats with respect to SBP were considered baroreceptor reflex sequences³⁸. The baroreceptor reflex sensitivity (BRS, ms•mmHg−1) was calculated as a linear regression coefficient averaged over all identified sequences (PI = BRS•SBP + const., where fitting of the curve was performed via the least squares method).

Statistical analysis (Hemodynamic data)

Hemodynamic data are presented as the means ± sds. The data were analyzed via GraphPad Prism software (version 8.4.2). The Shapiro‒Wilk test was used to assess normality. Cardiovascular parameters were compared via two-factor ANOVA to evaluate the main effects of strain, sex, and their interaction. A t test for unpaired observation was applied to compare intra- and intergroup differences. A p value < 0.05 was considered statistically significant.

PVN Sampling

Using a cryostat (Leica Microsystems CM1900, Leica Microsystems, Nussloch GmbH, Nussloch, Germany), caudal-to-rostral brain sections of 60 μm thickness were taken and stained with toluidine blue (1% [v/v] in 70% [v/v] ethanol, Sigma‒Aldrich Co. Ltd., Poole, Dorset, UK) for mapping and verification of the hypothalamic PVN. Unstained tissue was then bilaterally punctured, and 1 mm diameter tissue samples were obtained via a puncher with a 15-G microsampling needle (Sample Corer, Fine Science Tools Inc., Foster City, CA, USA; cat. no. 18035-01). The sampled PVN tissue was stored in RNAse-free tubes (ISOLAB Laborerate GmbH, Eschau, Germany) at -80°C.

RNA Extraction and Sequencing

RNA extraction and library construction were performed as described previously³⁹. Four groups of animals were used: Wistar male rats (n=4), Wistar female rats (n=5), SHR males (n=5), and SHR females (n=5). The samples were lysed via the QIAzol lysis reagent (Qiagen), and total RNA was extracted via the Direct-zol RNA MiniPrep extraction kit (Zymo Research, Irvine, CA, USA). The RNA integrity number (RIN) was determined via an Agilent 2200 TapeStation system (Agilent Technologies, 2503). Samples with a RIN >8 were used for sequencing. cDNA libraries were prepared via the TruSeq® Stranded mRNA Library Prep (Illumina, 20020594) and sequenced via the NextSeq500 High Output Version 2.5, 2 × 75 bp kit (Illumina, 15057931) at Bristol Genomics Facility, University of Bristol. Each sample generated >35 million PE reads, according to MultiQC⁴⁰.

RNA-seq data analysis

Paired-end sequencing data (FASTQ) generated on the Illumina platform were first processed via a trimming step (GSE:309435). This preprocessing included adaptor clipping, quality-based filtering, and length selection, carried out with Trimmomatic⁴¹, using a Phred score threshold of 30 (≈99.9% base call accuracy). Posttrimming quality assessment was performed with MultiQC⁴⁰^, and the resulting high-quality reads were then aligned to the Rattus norvegicus reference genome (Rn7) using the Spliced Transcripts Alignment to a Reference (STAR) aligner⁴². The counts of aligned reads were calculated using FeatureCountsvia featureCounts⁴³.

Statistical analyses were conducted in the R programming language (version 4.4.1)⁴⁴. The data were first normalized via the median-of-ratios method⁴⁵ built into DESeq2 (version 1.44.0)⁴⁶, which enabled us to confidently predict differentially expressed genes (DEGs). Only DEGs with adjusted P values (P adjusted) < 0.05 were considered significant. Gene annotation was performed via the ClusterProfiler package (version 4.10.1)⁴⁷, which retrieves genome annotations from ENSEMBL⁴⁸. For correlation and linear regression analyses, we applied the Spearman method from the Hmisc package (version 5.1--3) and the Stats package (version 4.3.1), respectively. The data were visualized via custom scripts written in R via the “ggplot2” (v3.5) package.

Functional classification and gene ontology

To explore the functional profiles of the DEGs, we organized the genes according to their physiological and pharmacological roles. Gene classification was carried out following the categories proposed by The International Union of Basic and Clinical Pharmacology (IUPHAR) system⁴⁹, which groups genes into categories such as endogenous peptides, G protein-coupled receptors, catalytic receptors, enzymes, channels, transporters, and other pharmacological targets. In addition, we defined an extra category specifically for transcription factors that have been previously described in the human literature⁵⁰.

To investigate the pathways modulated by the DEGs, we performed functional enrichment analyses via Gene Ontology (GO) and Reactome, implemented through the Cluster Profiler (version 4.10.1)⁴⁷and Reactome (version 1.48)⁵¹ packages. These analyses identified pathways in the biological process (BP), molecular function (MF), and cellular component (CC) categories and the KEGG and Reactome categories^51–53. To use these tools, the first step was to convert the gene symbols to Entrez IDs via the ClusterProfiler package⁴⁷, which uses only genes with a BaseMean ≥ 5 as the background for overrepresentation analysis. The analyses were conducted with the org.Rn.eg.db database (version 3.18.0). Pathways were considered significantly altered when the P. Adjusted were < 0.05.

Hemodynamic parameters and autonomic portrayal of WR and SHR males and females

Physiological analysis revealed significant differences in cardiovascular parameters between SHRs and WRs, with distinct profiles for males and females. For SBP, two-way ANOVA revealed significant main effects of strain (F = 215.509, P ≤ 0.0001) and a significant strain-by-sex interaction (F = 5.385, P = 0.032) (Fig. 1A). This pattern extends to SBP variability, with significant strain effects and interactions for total variability (TV-SBP: F = 20,346 P ≤ 0,0001; F = 12,135 P = 0,002), very-low-frequency SBP (VLF-SBP: F = 11,157 P = 0,003; F = 10,132 P = 0,005) and low-frequency SBP (LF-SBP: F = 5,5741 P = 0,029; F = 11,458 P = 0,003) (Fig. 1B-D). High-frequency SBP (HF-SBP) analysis revealed only a strain effect (F = 35,6 P ≤ 0,0001) (Fig. 1E). Post hoc tests confirmed that SBP was greater in SHRs than in WRs in both sexes. Notably, SHR males presented significantly increased TV-SBP and other variability metrics compared with those of WR males, whereas SHR females differed from WR females only in HF-SBP. Within-strain comparisons revealed that SHR females presented lower SBP, TV-SBP, and VLF-SBP than SHR males did, indicating a less severe autonomic phenotype in hypertensive females.

Similar patterns were observed for diastolic blood pressure (DBP), with significant strain and interaction effects on DBP (F = 65,404 P ≤ 0,0001; F = 5,275 P = 0,033), as well as in the variability analyses, with TV-DBP (F = 4,79 P = 0,041; F = 4,211 P = 0,05) and VLF-DBP (F = 4,781 P = 0,041; F = 4,052 P = 0,05) (Supplementary Fig. 1A-C). Strain vs. sex effects on LF-DBP were also observed (F = 7,650 P ≤ 0,012; F = 6,693 P = 0,018) (Supplementary Fig. 1D). Strain alone influenced HF-SBP (F = 16,603 P = 0,001) (Supplementary Fig. 1E). Intergroup comparisons revealed higher DBP, TV-DBP, VLF-DBP, LF-DBP and HF-DBP in SHR males than in WR males. In SHR females, DBP and HF-DBP were greater than they were in WR females. Intragroup differences indicate greater normal DBP, TV-DBP, and LF-DBP in WR females than in males, whereas no significant difference in DBP or its variability was detected between male and female SHRs.

We also analyzed heart rate (HR), which demonstrated a significant sex effect (F = 9.611, P = 0.006) (Supplementary Fig. 2A), where intergroup comparisons revealed higher HRs in SHRs than in males. Furthermore, in terms of variability analyses, we observed a strain effect on TV-HR (F = 4.789, P = 0.041) (Supplementary Fig. 2B) and VLF-HR (F = 5.219, P = 0.034) (Supplementary Fig. 2C) and a sex effect on LF-HR (F = 7.743, P = 0.012) (Supplementary Fig. 2D). For HF-HR, no significant effects of sex or strain were observed (Supplementary Fig. 2E). However, our analyses of the LF/HF HR ratio (Supplementary Fig. 2F) revealed significant effects of both sex (F = 6.912, P = 0.017) and strain (F = 6.648, P = 0.018). Finally, the BRS analysis revealed no significant effects of strain, sex, or their interaction (F = 2.630, P = 0.121; F = 0.570, P = 0.460; F = 0.052, P = 0.821; data not shown).

Characterization of the PVN transcriptomic profile in both sexes

RNA sequencing of the PVN identified over 21,000 expressed genes, with a core set of ~ 17,400 genes with a mean expression greater than 5 retained for differential analysis. These genes are distributed across all chromosomes in males and females (Supplementary Fig. 3A and B, Supplementary Table 1), including their median expression. Next, we categorized the genes on the basis of IUPHAR and TF classification and selected those with the highest expression levels in both males and females. Our analysis revealed strong conservation of expressed genes between the sexes and several expected genes, such as those in the endogenous peptide classification, including Oxytocin (Oxt), Vasopressin (Avp), and Angiotensinogen (Agt), in male (Supplementary Fig. 3C, Supplementary Table 2) and female rats (Supplementary Fig. 3D, Supplementary Table 2).

Sex differences in the PVN transcriptome in normotensive and hypertensive rats

We observed strong conservation among the genes identified and expressed in each sex and strain (Fig. 2A). Further analysis of the base mean counts in males and females revealed a strong correlation between gene expression in WR and SHR (males: r = 0.994, slope = 1.116; females: r = 0.994, slope = 0.942). However, a significant difference in the regression slopes was observed (p < 0.0001; Fig. 2B). While both sexes presented highly correlated gene expression patterns between SHR and WR, the males presented a greater increase in gene expression in the SHR strain (slope above 1), with females showing a slight decrease in gene expression (slope below 1). This could indicate sex-specific differences in how gene expression is regulated between the two strains. (Fig. 2B).

To better understand the impact of sex on the gene expression profiles of the two strains, we conducted a differential expression analysis. Principal Component Analysis (PCA) revealed that the samples clustered distinctly based on both strain and sex, confirming the strong dimorphism of the PVN transcriptomic profile (Fig. 2C). Delving deeper into the impact of sex on the PVN transcriptome, we found a modest effect in normotensive rats, with only 18 DEGs between WR female and male rats (8 upregulated, 10 downregulated) (Fig. 2D, Supplementary Table 3). In contrast, the difference was amplified in the SHR strain, with 311 DEGs between SHR females and males (168 upregulated, 143 downregulated) (Fig. 2E, Supplementary Table 4). Among these DEGs, 9 were commonly regulated in both strains, primarily those related to sex chromosomes (Fig. 2F).

The 10 upregulated genes in the PVN of female Wistar rats revealed GO terms related to the receptor complex (GO:0043235), interleukins (GO:0070671, GO:0032729, GO:0032609, GO:0032649, rno04060, R-RNO-447115, and R-RNO-449147), and collagen processes (GO:0032967, GO:0010714, GO:0032965, GO:0010712, GO:0030199, GO:0032964, and GO:0032963). Conversely, pathways associated with the 8 downregulated genes in the PVN of Wistar females and consequently enriched in males were associated with potassium channels (GO:0005242, R-RNO-1296041, and R-RNO-1296059), ligand-gated channel activities (GO:0005217, GO:0015276, GO:0022834, GO:0099094, and GO:0099604), and the oxytocin signaling pathway (rno04921) (Fig. 2G, Supplementary Table 5).

In the SHR strain, pathways enriched with genes upregulated in females revealed mechanisms related to RNA polymerase II (R-RNO-73856), the extracellular matrix and collagen organization (GO:0031012, GO:0062023, GO:0030020, GO:0005201, R-RNO-1474244, GO:0005581, GO:0098644, GO:0005583, GO:0098643, R-RNO-8948216, R-RNO-1650814, and R-RNO-1474290). However, pathways related to genes downregulated in females and consequently upregulated in males included nervous system processes involved in the regulation of systemic arterial blood pressure (GO:0001976), neurodegeneration (rno05012) and protein folding (GO:0006457 and GO:0061077) (Fig. 2H, Supplementary Table 6).

The nine genes commonly regulated by sex in both strains were related to the sex chromosomes, with six genes associated with the X chromosome, including DEAD-Box Helicase 3 X-Linked (Ddx3x), Eukaryotic Translation Initiation Factor 2 Subunit Gamma (Eif2s3), Lysine Demethylase 6A (Kdm6a), Polysaccharide Biosynthesis Domain Containing 1 (Pbdc1), and the pseudogenes ENSRNOG00000037911 and ENSRNOG00000065796. Moreover, three genes related to the Y chromosome were observed: DEAD-Box Helicase 3 Y-Linked (Ddx3/Ddx3y/ENSRNOG00000057231), Lysine Demethylase 5D (Kdm5d), and Eukaryotic Translation Initiation Factor 2 Subunit 3, Structural Gene Y-Linked (Eif2s3y). These common genes demonstrated a conserved alteration influenced by sex, with those enriched in females related to translational initiation (R-RNO-72737, R-RNO-72649, R-RNO-72613, GO:0031369, GO:0005852, and GO:0003743), chromatin DNA binding (GO:0031490), and the histone complex (GO:0141052, GO:0035097, and GO:0032452). In contrast, those enriched in males also influenced pathways related to translational initiation (GO:0002183, GO:0006413, GO:0003743, GO:0008135, and GO:0090079), histone complexes (GO:0141052, GO:0032452, and R-RNO-3214842) and steroid hormone receptors (GO:0030521, GO:0060765, GO:0050681, GO:0030518, and GO:0033143) (Fig. 2I, Supplementary Table 7).

Transcriptomic Profile of Hypertensive Males Reveals Sympathoexcitatory and Stress Pathways

Transcriptomic differences between strains in male rats were clear in the PCA (Fig. 3A). A comparison of SHR males with WR males revealed a massive transcriptomic shift, with 3,013 DEGs (1,335 upregulated and 1,678 downregulated) (Fig. 3B, C and Supplementary Table 8). The categorization of these DEGs by the IUPHAR and TF databases revealed some potentially important genes for PVN functionality. The upregulated genes, including the endogenous peptides Hypocretin Neuropeptide Precursor (Hcrt) and Brain-Derived Neurotrophic Factor (Bdnf), are key drivers of sympathetic activation. In the GPCR category, genes such as Angiotensin II Receptor Type 1 (Agtr1a), Atypical Chemokine Receptor 3 (Ackr3), and the catalytic receptor TNF Receptor Superfamily Member 12A (Tnfrsf12a) were also upregulated (Fig. 3D, Supplementary Table 8). The differentially expressed genes also revealed important gene ontology terms, such as the upregulated genes, which were related to the cellular response to stress processes (R-RNO-2262752 and GO:0034976), protein folding (GO:0006457, GO:0051082, GO:0061077, GO:0051087, GO:0044183, GO:0140662, GO:0101031, GO:0006458, GO:0051084, GO:0051085, and GO:0042026), protein localization (GO:1903405, GO:1990173, GO:0034504, GO:1904851, GO:1900182, GO:0070203, GO:0070202, GO:0072594, GO:1900180, GO:1904816, and GO:0070200), and transcription complexes (GO:0090575). Conversely, the downregulated genes were associated with processes involving the extracellular matrix (GO:0031012, GO:0062023, GO:0030020, and GO:0005201) and gas transport (GO:0015669, GO:0005344, and GO:0015670) (Fig. 3E, Supplementary Table 9).

Transcriptomic changes in the PVN of hypertensive female rats

The clear effect of strain on the PVN of female rats was also evident, as demonstrated by PCA (Fig. 4A). A comparison of SHR females with WR females revealed 1,122 DEGs (435 upregulated, 687 downregulated) (Fig. 4B, C and Supplementary Table 10). The categorization of these DEGs by the IUPHAR and TF databases revealed several potentially important genes that were differentially expressed in female SHRs compared with Wistars. Specifically, among the upregulated genes, the NLR Family CARD Domain Containing 3 (Nlrc3) was noted in the catalytic receptor category, whereas the downregulation of Absent in Melanoma 2 (Aim2) was observed in the other protein category (Fig. 4D, Supplementary Table 10). The impact of these transcriptomic alterations was also reflected in the gene ontology analyses, revealing pathways related to myelination (GO:0042552, GO:0022010, GO:0043209, GO:0043218, and GO:0019911), neuron ensheathment (GO:0032291, GO:0007272, and GO:0008366), and the regulation of glial cells (GO:0045685, GO:0010001, GO:0097386, GO:0021782, GO:0048713, GO:0014003, and GO:0048709) related to the downregulated genes. Moreover, pathways related to the axoneme (GO:0005930), ciliary plasm (GO:0097014), and plasma membrane-bound cell projection cytoplasm (GO:0032838) were enriched with the upregulated genes (Fig. 4E, Supplementary Table 11).

Common and unique genes affected by hypertension in the PVN of male and female rats

Compared with those in WRs, the transcriptomic changes in the PVN of SHRs were notably more pronounced in males than in females. Among the DEGs, 2,458 DEGs were uniquely affected in males, and 567 were uniquely affected in females, according to the blood pressure state of each sex, with 555 genes commonly affected in both sexes (Fig. 5A). A distribution analysis of the commonly regulated genes in both sexes revealed that 552 (99.45%) changed in the same direction and were significantly positively correlated between males and females (R = 0.942, p < 0.0001). Among these genes, several were upregulated in both males and females, such as Serum/Glucocorticoid-Regulated Kinase 1 (Sgk1) and Ly6/PLAUR domain containing 10 (Lypd10), whereas E2F Transcription Factor 2 (E2f2) and Amylase Alpha 1A (Amy1) were downregulated regardless of sex. Among the commonly regulated genes, three were significantly regulated in both sexes but in opposite directions: Mannose Receptor C-Type 1 (Mrc1) and Potassium Inwardly Rectifying Channel Subfamily J Member 4 (Kcnj4) were downregulated in males and upregulated in females, whereas Adipocyte-related X-chromosome expressed sequence 2 (Arxes2) was upregulated in males and downregulated in females (Fig. 5B, Supplementary Table 12). Furthermore, linear regression analysis comparing the effects of common genes on male and female DEGs (Fig. 5C) revealed that females presented a greater coefficient of determination (males: r = 0.363, R² = 0.107, slope = 0.042; females: r = 0.412, R² = 0.184, slope = 0.058), indicating a significant difference in slope (F = 17.17, p < 0.001) and indicating a stronger transcriptomic response to hypertension in females than in males.

The classification of these common genes by the IUPHAR and TF databases revealed potentially important genes regulated by the SHR strain in each category. For example, in the enzyme category, there was an upregulation of the zeta chain of T-cell receptor-associated protein kinase 70 (Zap70) and Sgk1, whereas a downregulation of the endogenous peptide Annexin A1 (Anxa1) was observed (Fig. 5D, Supplementary Table 12). Among the pathways commonly affected by the hypertensive strain in male and female rats, six pathways were conserved (Fig. 5E). These included pathways related to cytoplasmic projection (GO:0032838), ciliary plasm (GO:0097014), and axoneme (GO:0005930) due to the positively regulated genes, whereas the negatively regulated genes affected gas transport pathways (GO:0015669, GO:0015670, and GO:0005344) (Fig. 5F, Supplementary Table 13).

Associations between hypertension and sex differences in the PVN transcriptome

To identify key sex-specific drivers of hypertension, we correlated genes differentially expressed by strain with those differentially expressed by sex in the SHR groups. This analysis revealed 146 genes in common (Fig. 6A). A correlation analysis of these 146 genes revealed a strong and significant correlation (r = 0.901, p = 0.0001), with 35 genes being positively regulated in males and associated with the hypertensive strain, including Adrenoceptor Alpha 2A (Adra2a), Angiotensin II Receptor Type 1 (Agtr1a), Regulator Of G Protein Signaling 2 (Rgs2), TNF Receptor Superfamily Member 12A (Tnfrsf12a), Atypical Chemokine Receptor 3 (Ackr3), and Brain-Derived Neurotrophic Factor (Bdnf) (Fig. 6B, Supplementary Table 14). The positively regulated genes were associated with steroid hormone receptor signaling (GO:0031958, GO:0071383, GO:0033143, GO:0030518, GO:0043401, and GO:0033145), nervous system processes involved in the regulation of systemic arterial BP (GO:0001976), and G protein regulation (GO:0045744, GO:0008277, GO:0001637, and GO:0004930). In contrast, the downregulated genes were associated with processes involving the extracellular matrix process (R-RNO-1474228 and R-RNO-216083) (Fig. 7C, Supplementary Table 15).

Moreover, in the female analysis, among the genes regulated by the hypertensive strain in females and those affected by sex in the SHR lineage, 36 genes that were commonly associated were identified (Fig. 6D), revealing a strong and significant correlation (r = 0.792, p = 0.0001). Among these genes, 11 were positively enriched in females and in the hypertensive strain; notably, they included Nlrc3 and Paired Ig-like Receptor B (Pirb) (Fig. 6E, Supplementary Table 14). The positively regulated genes were shown to be related to specific pathways, such as neutrophil degranulation (R-RNO-6798695), whereas the negatively regulated genes influenced pathways associated with neurodegeneration (rno05022), the ribosomal complex (GO:0000028), and glial cell projection (GO:0097386) (Fig. 6F, Supplementary Table 16).

This study provides, for the first time, a comprehensive transcriptomic catalog of the PVN in male and female normotensive and hypertensive rats, revealing profound sexual dimorphism in the molecular pathways involved in the development of hypertension. Our findings demonstrate that, in males, hypertension is associated with a robust transcriptomic signature of heightened sympathetic activity and neuroinflammation, whereas females exhibit a distinct profile suggestive of protective, anti-inflammatory adaptations. These molecular data align with our physiological findings of heightened vascular sympathetic outflow in SHR males compared with females; the SHR female autonomic profile is indicative of better prognosis and supports the finding of the PVN transcriptome.

Our autonomic analysis revealed significant cardiovascular differences driven by both hypertensive strain and sex, providing a physiological foundation for our transcriptomic findings. As anticipated, SHR of both sexes had significantly elevated blood pressure and increased blood pressure variability across all frequency bands (TV-BP, VLF-BP, LF-BP, and HF-BP) compared with their normotensive Wistar counterparts. The increase in BP variability in SHRs compared with WRs has been described previously^54–58. However, other authors found no evidence to support increased BP variability in SHRs⁵⁹. This difference in findings may be attributed to the different ages of the SHRs used in the different experiments. It is acknowledged that older SHRs often develop arterial stiffening, which can mask or even attenuate these fluctuations^55,60–62. The young 12-week-old rats used in our study were in a phase of established but not advanced hypertension. Therefore, the observed increases in VLF-BP and LF-BP variability are clear physiological indicators of heightened activation of both the RAS and sympathetic outflow directed to the vasculature ^34,63,64. We also found increased HF-BP variability in SHRs. HF-SBP arises from respiration-induced perturbations in cardiovascular hemodynamics in great thoracic blood vessels. These HF BP oscillations can be enhanced by vessel unloading⁶⁴ or slow breathing patterns⁶⁵. In our experiment, the SHR did not exhibit changes in breathing frequency, as judged by the position of the HF peak in both the SBP and HR spectra (data not shown). It is possible that the somewhat smaller circulating volume in SHRs than in WRs⁶⁵ could be reflected in the higher HF-SBP in SHRs.

Interestingly, the LF/HF HR ratio was lower in SHRs of both sexes than in Wistar rats. This suggests the activation of compensatory cardiac vagal mechanisms that counteract chronic BP elevation in the early phase of hypertension when BRS is still preserved in 12-week-old SHRs. In advanced stages of hypertension, the BRS decreases and predicts hypertension complications⁶⁶. Crucially, we also observed profound sexual dimorphism within the hypertensive group. SHR females had lower BP and TV BP and VLF-BP than males did. This more favorable cardiovascular profile in females is possibly due to the well-documented estrogen-mediated protective effects on blood vessels, better vascular compliance⁶⁷, and upregulation of vascular endothelial β-adrenoceptor expression, which mediates vasodilation and reduces the reactivity of vasoconstrictors⁶⁸. Furthermore, the lower heart rate in SHR females than in males suggests more effective cardiac vagal control and consequently better regulation of blood pressure, reinforcing the conclusion that females exhibit a less severe hypertensive phenotype at this age.

The sexual dimorphism in hypertension is well documented in humans, with men facing a greater risk than women do, particularly in early adulthood. Our transcriptomic analysis of the PVN seems to mirror this clinical reality at the molecular level, as SHR males presented a far greater number of DEGs than females did. PCA revealed that the samples clustered distinctly on the basis of these two factors, with sexual dimorphism becoming dramatically more pronounced under hypertensive conditions. In the normotensive WR strain, the differences between sexes are modest, with DEGs associated with inflammasome activity in females, whereas in males, enrichment in the GABAergic, GPCR, and oxytocin signaling pathways is detected. However, in the hypertensive SHR strain, these differences were profound. The female hypertensive profile is characterized by changes in the extracellular matrix, whereas the male profile is dominated by a massive upregulation of pathways central to blood pressure regulation, protein folding and secretion, as well as the emergence of pathways related to neurodegeneration, a condition strongly correlated with hypertension in the literature^69,70. These divergent molecular signatures strongly suggest that the central mechanisms driving hypertension in the PVN are fundamentally different between males and females.

The transcriptomic disruption in male SHRs is characterized by a clear prohypertensive signature, including key genes, such as the upregulation of the endogenous peptides Hcrt, Bdnf, and Calca. These genes are extremely relevant in the PVN because of their role in increasing sympathetic tone^71–74. This sympathoexcitatory drive is further amplified by the increased expression of the critical GPCR Agtr1a (the Angiotensin II Type 1a receptor). This gene has already been linked to blood pressure regulation by reducing inhibitory GABAergic input and increasing excitatory glutamatergic input to PVN neurons, thereby enhancing sympathetic outflow^75–78. The upregulation of these genes provides a direct molecular explanation for the increased VLF-SBP and LF-SBP (hallmarks of sympathetic and RAS activation) observed in male SHRs compared with normotensive strain. This prohypertensive state is compounded by neuroinflammation ^79,80, as evidenced by the increased expression of genes such as the GPCR Ackr3 ⁸¹. The broad upregulation of pathways related to protein transcription, folding, and cellular stress likely reflects the high metabolic demand placed on PVN neurons by this state of chronic sympathoexcitation and neuroinflammation^78,79.

In contrast to males, females presented a smaller change in the transcriptomic profile within their PVN, which was more constrained and pointed toward a protective, anti-inflammatory response. Among the DEGs, the downregulated gene Anxa1 plays important roles in the functionality of the nucleus because of its involvement in preventing neuroinflammation⁸². The decrease in the expression of this gene is consequently associated with a proinflammatory state. Most notably, we observed the coordinated regulation of two key inflammasome components: the upregulation of the catalytic receptor Nlrc3, which is known to inhibit inflammasome activity ^83–85, and the concurrent downregulation of Aim2, a sensor protein that initiates inflammasome assembly⁸⁶. The simultaneous upregulation of an inhibitor and downregulation of an activator strongly suggests a concerted, multipronged mechanism designed to actively suppress neuroinflammation in the female PVN. This active immunomodulation likely explains pathways related to the neuronal ensheathment, myelination, and glial cell differentiation observed in females. We propose that compensatory anti-inflammatory mechanisms are key factors in producing the milder hypertensive phenotype in females, as reflected by their lower blood pressure than in males.

Despite the profound sex-specific differences, 555 genes were commonly regulated by hypertension in both sexes, revealing a core pathological response. These shared genes constituted a smaller fraction of the total DEGs in males but accounted for more than half of the changes in females, underscoring the highly distinct mechanisms driving hypertension in males. Among these common genes, there was remarkable conservation in the direction of alteration between sexes, as demonstrated by the strong correlation in their expression. However, females presented a stronger transcriptomic response to hypertension than did males within this shared gene set. Central to this shared signature is a proinflammatory state driven by the downregulation of Anxa1, encoding an important anti-inflammatory peptide⁸², which is reduced in both sexes, driving a proinflammatory basal state likely contributing to neuroinflammation, an important condition associated with hypertension. This finding is further supported by evidence of glial dysfunction, as indicated by the downregulation of Aqp4, an ion channel crucial for astrocyte functionality in the hypothalamus, indicating dysfunction in this type of cell functionality⁸⁷. Another important upregulated common gene was Sgk1, an enzyme-linked to cellular stress and death, which indicates a negative feedback mechanism involving neurotrophic factors such as BDNF, leading to decreased neuronal survival and function⁸⁸. Together, these findings suggest that hypertension establishes a common foundation of neuroinflammation and glial dysfunction in the PVN, upon which more dramatic and sex-specific mechanisms are built.

To better understand the sex-specific impact of the PVN in driving hypertension, we correlated the genes differentially regulated by sex in SHRs with those influenced by hypertension. This analysis powerfully isolated the distinct molecular drivers responsible for the divergent paths of males and females. In males, this approach highlighted genes such as Agtr1a and Rgs2 as central to both the hypertensive state (upregulated in SHRs vs. Wistar) and the more severe male phenotype (higher expression in SHR males than in SHR females). Recent findings have shown that the overexpression of Rgs2 in the PVN is associated with the attenuation of blood pressure increases and sympathetic outflow caused by angiotensin II and its receptor, likely acting as a compensatory mechanism to control hypertension⁸⁹. These mechanisms affect the RAS pathway, suggesting a possible mechanism underlying the VLF-SBP increase observed in SHR males in relation to females. Additionally, a positive correlation between the gene expression of males and the SHR strain was observed in the expression of Bdnf, encoding a peptide involved in the activation of sympathetic tone⁷³. On the other hand, in females, there was an increase in the expression of Nlrc3 and Pirb, which act in anti-inflammatory pathways^83–85,90. These findings suggest that these anti-inflammatory genes are key drivers of the protective female phenotype, likely acting as compensatory mechanisms to reduce neuroinflammation, which is a hallmark of the hypertensive PVN.

While this study provides a comprehensive transcriptomic overview, some limitations must be acknowledged. We used bulk RNA sequencing, which allowed us to obtain a general overview of the transcriptomic effects of sex and hypertension in the PVN. However, these methods cannot resolve which specific cell populations (neurons, astrocytes, or microglia) are responsible for the observed changes. While our data reveal strong correlations, they cannot define whether these transcriptomic changes are a cause or a consequence of hypertension.

In summary, our results reveal sexual dimorphism in the transcriptomic landscape of the PVN in hypertension. However, a pronounced sex-specific effect was observed in the mechanisms driving hypertension in each sex. The spectral analysis of SBP revealed a striking increase in sympathetic outflow, predominantly in males, a finding that is molecularly substantiated by the male-exclusive upregulation of prohypertensive genes such as Calca, Bdnf, Hcrt, and Agtr1a. In addition to these prohypertensive genes, the upregulation of Rgs2 in males, compared with females, suggests a compensatory mechanism to block the angiotensin II cascade. In contrast, the SHR females presented expression patterns related to anti-inflammatory pathways, such as the downregulation of Aim2 and the upregulation of Nlrc3 and Pirb, which occurred exclusively in females. This molecular profile likely explains the lower prevalence and severity of hypertension in females, driven by the active suppression of genes involved in inflammatory pathways and the absence of the sympathoexcitatory gene signature observed in males.

In conclusion, our analyses provide new insights into the cardiovascular and molecular basis of sex differences in hypertension. We identified the presence of a neuroinflammatory mechanism in the PVN, suggesting a strong contribution to the hypertensive state. Our results also revealed a greater contribution of sympathetic tone in males, whereas in females, a stronger anti-inflammatory component was observed, which may explain the lower prevalence and severity of hypertension in females. These findings offer a valuable framework for the development of future sex-specific pharmacotherapeutic strategies.

Perspectives and significance

Our current study revealed several mechanisms contributing to the hypertensive state, as well as compensatory mechanisms across sexes. Our analyses provide new insights into the impact of hypertension on the PVN and highlight mechanisms that could be targeted to control blood pressure. Future transcriptomic studies will expand these analyses to the cellular level, for example, through single-cell RNA sequencing (scRNA-seq), and to other key brain regions, such as the rostral ventrolateral medulla (RVLM), supraoptic nucleus (SON), and nucleus tractus solitarius (NTS), offering a broader perspective on the role of the brain in hypertension and how these areas are affected.

Acknowledgments

The authors are grateful to MsC. Marcelo Henrique Sales for his technical assistance.

Author contributions

V.J.D.: Methodology, Investigation, formal analysis, data curation, writing original draft, writing review & editing.

J.V.N.: Formal analysis, writing original draft, writing review & editing.

M.J: Investigation, formal analysis, writing original draft, writing review & editing.

M.L.: Investigation, writing review & editing.

O. Š.: Investigation, writing review & editing.

A.G.P.: Investigation, Writing review & editing.

D.M.: Conceptualization, funding acquisition, writing review & editing.

N.J.Ž.: Conceptualization, funding acquisition, investigation, project administration, writing original draft, writing review & editing.

A.S.M.: Conceptualization, funding acquisition, methodology, project administration, formal analysis, writing original draft, writing review & editing.

Funding

V.J.D, J.V.N, A.S.M.: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [Nos. 2024/15733-8, 2021/14426-6, 2019/27581-0, 2024/01620-7 and 2024/19300-9], Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) – Finance Code 001 and National Council for Scientific and Technological Development (CNPq) [Nos. 421434/2023-6 and 402719/2022-0). M.J., N.J-Ž, M.L.: Ministry of Science, Technological Development and Innovation of Serbia, grant number 451-03-137/2025-03/200110. DM.: Medical Research Council (Grants MR/W028999/1 and MR/N022807/1) and Biotechnology and Biological Sciences Research Council (BB/R016879/1).

Availability of data and materials

In addition to the data reported in the manuscript and additional files, all the raw data files are available through the NCBI Gene Expression Omnibus (GEO) platform (GSE: GSE309435).

Ethics approval and consent to participate

These experiments were approved by the Ethics Review Board of the School of Medicine, University of Belgrade, and the relevant ministry of the Republic of Serbia (license no. 119–01–13/17/2015–09). Furthermore, all studies involving animals were conducted in compliance with the ARRIVE guidelines for reporting animal experiments.

Consent for publication

Not applicable.

Competing interests

The authors have no competing interests to declare.

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Transcriptomic Profiling of the Hypothalamic PVN Reveals Sexually Dimorphic Pathways of Sympathoexcitation and Neuroinflammation in a Rat Model of Hypertension

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Version 1

Abstract

Figures

Plain English Summary

Highlights

Background

Methods

Statistical analysis (Hemodynamic data)

Functional classification and gene ontology

Results

Hemodynamic parameters and autonomic portrayal of WR and SHR males and females

Characterization of the PVN transcriptomic profile in both sexes

Sex differences in the PVN transcriptome in normotensive and hypertensive rats

Transcriptomic Profile of Hypertensive Males Reveals Sympathoexcitatory and Stress Pathways

Transcriptomic changes in the PVN of hypertensive female rats

Common and unique genes affected by hypertension in the PVN of male and female rats

Associations between hypertension and sex differences in the PVN transcriptome

Discussion

Conclusion

Declarations

Acknowledgments

Author contributions

Funding

Availability of data and materials

References

Additional Declarations

Supplementary Files

Status:

Version 1