This study was conducted in accordance with protocols approved by the ethics committee of The Eighth Affiliated Hospital of Sun Yat-sen University (Shenzhen, China). Following informed consent, serum samples were procured from 32 patients diagnosed with hypertension and 30 normotensive, healthy volunteers. The collected serum was utilized to quantify the concentrations of FGF1, VEGFA, ANGPT1, and AGGF1 using commercial ELISA kits. A comprehensive summary of the participants' clinical characteristics is detailed in Supplementary Table 1.

All animal experiments were performed under the ethical guidelines and approval of the Animal Care and Use Committees at The Eighth Affiliated Hospital of Sun Yat-sen University. To induce hypertension, C57BL/6J mice were subjected to a continuous 4-week subcutaneous infusion of Angiotensin II (AngII, Sigma-Aldrich) at a rate of 0.8 mg/kg/day, delivered via surgically implanted osmotic mini-pumps. A control group received saline infusions under identical conditions. Systolic blood pressure was assessed throughout the study using a Softron BP-2010A noninvasive tail-cuff system.

All animal experiments were conducted in accordance with institutional guidelines and approved protocols for the care and use of laboratory animals.

Animals were anesthetized using isoflurane inhalation anesthesia. Anesthesia was induced with 4% isoflurane and maintained at 1%–2% isoflurane delivered in oxygen using an anesthesia machine. Adequate depth of anesthesia was confirmed by the absence of pedal withdrawal reflex prior to experimental procedures.

At the end of the experiment, animals were humanely euthanized by an overdose of anesthetic, followed by confirmation of death through cessation of respiration and heartbeat. All efforts were made to minimize animal suffering.

For endothelial-specific gene knockdown, 8-week-old male C57BL/6J mice received a single tail vein injection of recombinant AAV9 vectors (5 × 10¹¹ viral genomes/mouse) encoding either a BACH1-targeting siRNA (5'- GGAUCUACUACCAGGAGUATT -3') or a non-targeting scrambled control. One week after vector administration to allow for transduction, hypertension was induced with a 28-day AngII infusion (0.8 mg/kg/day). Blood pressure was assessed on a weekly basis, and at the study's conclusion, aortic tissues were harvested for subsequent morphological evaluation.

Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial-specific growth medium (ScienCell) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco). For stimulation experiments, HUVECs were treated with 10⁻⁶ M AngII (AmBeed) for 48 h.

Human 293 T cells were cultivated in DMEM containing 10% FBS and 1% penicillin-streptomycin. Both cell lines were maintained in a humidified incubator at 37 °C with 5% CO₂.

To assess arterial stiffness, aortic pulse wave velocity (aPWV) was measured in mice anesthetized with 2% isoflurane. With continuous ECG monitoring, Doppler probes were placed consecutively over the aortic arch and the abdominal aorta. The pulse transit time was derived from the interval between the ECG R-wave and the foot of the Doppler signal at each location. The aPWV was then calculated as the ratio of the distance between the probes to the differential transit time.

Aortic rings (2 mm) were excised from euthanized mice and mounted in a multi-wire myograph system (DMT620M) containing Krebs solution aerated with 5% CO₂ at 37 °C for isometric tension studies. After equilibration, the functional integrity of the rings was confirmed by their contractile response to 1 μM phenylephrine (PE) and subsequent endothelial-dependent relaxation with 30 μM acetylcholine (ACh). Cumulative concentration-response curves to the vasodilator sodium nitroprusside were subsequently generated. Vasorelaxation was expressed as the percentage reversal of the PE-induced pre-constriction.

Primary aortic endothelial cells (MAECs) were obtained from C57BL/6J mouse aortas using an explant culture technique. Aortic segments were placed lumen-side down on dishes coated with growth factor-reduced Matrigel (Corning) and cultured in ECM medium (Cellvita, 1010, Shanghai Medicell Biotechnology Co.,Ltd.) supplemented with growth factors and 10% FBS. After 4 days, endothelial cells that had migrated from the explants were expanded. The endothelial phenotype of the cultured MAECs was confirmed by assessing CD31 expression via flow cytometry.

Serum FGF1, VEGFA, ANGPT1 and AGGF1 concentrations were assessed by individual ELISA kits (R&D Systems, Abingdon, UK) following the manufacturer's instructions. Optical densities were recorded in a Multiskan GO spectrophotometer (Thermo Scientific, Waltham, USA) at 450 nm with a wavelength correction at 540 nm.

For in vitro gene silencing, siRNAs were prepared at a final concentration of 50 nmol/L. Transfection into cells was achieved using NanoTrans™ Transfection Reagent 3000 (CYTOCH), following the provided guidelines. Cells were collected at 48 h after transfection. The specific siRNA sequences are listed in Supplementary Table S2.

The angiogenic potential of HUVECs was assessed using a tube formation assay. Wells of a 96-well plate were coated with 80 μL of growth factor-reduced Matrigel and allowed to polymerize at 37 °C. HUVECs (1.5 × 10⁴ cells/well) were then seeded onto the Matrigel surface. After a 6-hour incubation period, the formation of capillary-like networks was documented with phase-contrast microscopy, and the total tubule length was quantified using the angiogenesis analysis tools in ImageJ.

Total RNA from cultured cells or tissue samples was isolated with TRIzol reagent (Invitrogen). Gene expression levels were quantified by RT-qPCR following manufacturer's protocols. A detailed list of primer sequences used for amplification is provided in Supplementary Table S3.

Protein lysates from cells and tissues were prepared using RIPA buffer (NCM, WB3100, China) containing a protease/phosphatase inhibitor cocktail and PMSF. Tissue samples were mechanically homogenized, while cell lysates were prepared by scraping. Protein concentration was determined using the BCA assay (Pierce™). Equal amounts of protein were resolved by SDS-PAGE, transferred to membranes, and probed with specific primary antibodies. Information regarding the antibodies is available in Supplementary Table S4.

To investigate transcriptional regulation, 293 T cells were co-transfected with a pGL4.10 vector containing the FGF1, VEGFA, ANGPT1 and AGGF1 promoter, a pcDNA3.1-BACH1 expression vector, and a pRL-TK Renilla luciferase control vector using Lipofectamine 3000 (Invitrogen). At 48 h post-transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase signals were normalized against the Renilla luciferase activity to correct for transfection efficiency.

ChIP assays were performed using the Pierce Magnetic ChIP Kit (Thermo Scientific) according to the supplier's protocol. Briefly, cells were cross-linked with formaldehyde, and chromatin was sonicated to an average size of ∼300 bp. The sheared chromatin was then subjected to immunoprecipitation overnight with antibodies against BACH1 or a non-specific IgG control. Following DNA elution and purification, enrichment of the FGF1, VEGFA, ANGPT1 and AGGF1 promoter region were quantified using SYBR Green-based qPCR. The primer sequences are detailed in Supplementary Table S5.

Paraffin-embedded mouse aortic sections were processed for immunofluorescence analysis. Following antigen retrieval and permeabilization, sections were blocked and then probed overnight at 4 °C with primary antibodies targeting BACH1. Staining was visualized using corresponding Alexa Fluor-conjugated secondary antibodies and DAPI for nuclear counterstaining. Images were acquired on a Zeiss LSM 880 confocal microscope, and fluorescence intensity analysis was conducted with ImageJ software.

Results are expressed as the mean ± standard deviation (SD) for continuous variables and as frequencies (%) for categorical data. For two-group comparisons, a two-tailed Student's t-test was utilized. For multi-group comparisons, one-way ANOVA followed by Tukey's post hoc tests was employed. Correlations between variables were evaluated using Pearson's coefficient. A P-value less than 0.05 was considered statistically significant. All statistical computations were performed using GraphPad Prism 9.0.

To investigate the impact of hypertension on angiogenesis, we established a hypertensive mouse model by continuous subcutaneous infusion of Angiotensin II (AngII, 0.8 mg/kg/day) via osmotic pumps for 4 weeks. Non-invasive monitoring confirmed a significant elevation in both systolic and diastolic blood pressure in the AngII-treated group relative to the saline-infused mice (Figures 1A–D). Furthermore, vascular function assessments revealed that AngII administration increased aortic pulse wave velocity (aPWV) and impaired vasorelaxation, indicative of significant vascular damage (Supplementary Figures S1A,B). To directly assess angiogenesis in vivo, we performed isolectin B4 (IB4) staining of retinal flat mounts. The results demonstrated a marked reduction in retinal vascular density in hypertensive mice (Figures 1E,F). These in vivo findings were corroborated by in vitro experiments, where AngII treatment significantly suppressed the tube formation capacity of endothelial cells (Figures 1G,H). Collectively, these data provided clear evidence from both in vivo and in vitro models that hypertension is characterized by deficient angiogenesis.

While the BACH family of transcription factors is known to play important roles in complex biological processes, including ischemic cardiovascular diseases, its potential involvement in hypertension has not been investigated. Therefore, we sought to determine whether BACH family members contribute to the pathogenesis of hypertension. Following treatment with AngII, we observed a specific and significant upregulation of BACH1 mRNA, whereas BACH2 transcript levels were unaffected in HUVECs (Figure 2A). This selective induction was further validated at the protein level, as demonstrated by western blotting assays (Figures 2B,C). Furthermore, immunofluorescence assays demonstrated that BACH1 is localized within the nucleus, and its intensity was markedly enhanced by AngII stimulation (Figures 2D,E).

Having established that BACH1 is upregulated by AngII stimulus, we next investigated its functional role in angiogenesis. We used small interfering RNA to knockdown BACH1 (Si-BACH1) in HUVECs, the efficiency was validated by qPCR and Western Blotting (Supplementary Figures S2A–C). Using an in vitro tube formation assay, we found that the genetic silence of BACH1 was sufficient to rescue endothelial cell angiogenic potential, leading to enhanced network formation (Figures 2F,G). These results identified BACH1 as a key factor upregulated in hypertension that suppresses angiogenesis, highlighting its potential as a therapeutic target for improving angiogenesis in hypertension.

Given that angiogenesis is regulated by a complex network of growth factors, we sought to identify the specific downstream targets through which BACH1 exerts its anti-angiogenic effects. Therefore, we performed series of key angiogenic regulators in endothelial cells following BACH1 inhibition. RT-qPCR analysis revealed that knockdown of BACH1 significantly upregulated the mRNA levels of Fibroblast Growth Factor 1 (FGF1), Vascular Endothelial Growth Factor A (VEGFA), Angiopoietin 1 (ANGPT1) and Angiogenic Factor with G-patch and FHA domains 1 (AGGF1). In contrast, the expression of other related factors, including FGF2, VEGFB, ANGPT2, PIGF, PDGFA and NOTCH1, remained unchanged (Figure 3A). This specific response was similarly found at the protein level using western blotting analysis (Figures 3B,C). These findings suggest that BACH1 negatively regulates angiogenesis primarily by suppressing the expression of FGF1, VEGFA, ANGPT1 and AGGF1. To validate the relevance of this axis in hypertensive condition, we treated HUVECs with AngII. As hypothesized, AngII stimulation induced a significant downregulation of FGF1, VEGFA, ANGPT1 and AGGF1 at both mRNA and protein levels (Figures 3D–F), furthermore, inhibition of BACH1 improves levels of FGF1, VEGFA, ANGPT1 and AGGF1 (Figures 3G–I).

Previous studies have established BACH1 as a key transcriptional regulator. We therefore questioned whether BACH1 directly inhibits the expression of these pro-angiogenic factors by binding on their regulatory regions. We performed Chromatin Immunoprecipitation followed by qPCR (ChIP-qPCR) in endothelial cells. The results demonstrated a significantly enhanced recruitment of BACH1 to the promoter regions of FGF1, VEGFA, ANGPT1 and AGGF1 in AngII-treated cells compared to the PBS control group (Figures 3J–M). To confirm this repressive interaction, we conducted dual-luciferase reporter assays, which showed that BACH1 overexpression inhibited the promoter activity of all four angiogenic factors (Figures 3N–Q).

Collectively, these mechanistic data provided evidence that BACH1 acts as a direct transcriptional repressor of FGF1, VEGFA, ANGPT1 and AGGF1. Under hypertensive conditions, elevated BACH1 levels lead to increased binding at these gene promoters, thereby suppressing their expression and culminating in the inhibition of angiogenesis.

To clarify the clinical relevance of FGF1, VEGFA, ANGPT1 and AGGF1 in hypertension, we enrolled 32 patients diagnosed with hypertension according to WHO criteria and 30 age-matched normotensive volunteers. Peripheral blood samples were collected, and the serum concentrations of the four angiogenic factors were quantified by ELISA. Consistent with our experimental data, the results revealed that circulating levels of FGF1, VEGFA and AGGF1 were significantly lower in hypertensive patients compared to healthy controls (Figures 4A–C). Although the concentration of ANGPT1 also showed a downward trend in the hypertensive group, this difference did not reach statistical significance (Figure 4D).

To further explore the relationship between these factors and hypertension, we performed a correlation analysis. A significant inverse correlation was observed between the circulating levels of all four factors—FGF1, VEGFA, ANGPT1 and AGGF1—and both systolic and diastolic blood pressure (Figures 4E–L). Notably, despite the lack of a significant difference in its mean concentration between the two groups, ANGPT1 levels still negatively correlated with blood pressure, consistent with the other factors.

Collectively, these clinical data demonstrated that in human, higher blood pressure is associated with lower circulating levels of key pro-angiogenic factors. This suggests that the FGF1, VEGFA, ANGPT1 and AGGF1 axis is dysregulated in hypertension and that interventions aimed at restoring the levels of these factors could represent a viable strategy to improve angiogenic function.

To definitively establish the regulatory role of BACH1 on pro-angiogenic factors in vivo, we employed an adeno-associated virus (AAV9) vector to achieve endothelium-enriched-knockdown of BACH1 (EC-KD) in C57BL/6J mice. Following successful vector delivery, these mice were subjected to AngII infusion to induce hypertensive models.

Remarkably, the selective depletion of endothelial BACH1 caused protection against the development of hypertension. Compared to hypertensive mice, endothelial BACH1 knockdown exhibited significantly attenuated systolic and diastolic blood pressure (Figures 5A–D). Furthermore, we assessed the impact on retinal microvascular using IB4 staining. This analysis revealed that inhibiting BACH1 expression successfully ameliorated the retinal vascular rarefaction characteristic of the hypertensive state (Figure 5E,F), indicating a restoration of angiogenic homeostasis.

To confirm the underlying molecular mechanism, we isolated primary aortic endothelial cells (MAECs) from these mice for further analysis. RT-qPCR results first validated the high efficiency of AAV9-mediated BACH1 knockdown in the target cells. Critically, in endothelial cells from the EC-KD group, the mRNA levels of FGF1, VEGFA, ANGPT1 and AGGF1 were significantly restored compared to the suppressed levels in the hypertensive group (Figure 5G). These findings were further corroborated at the protein level, as Western blot analysis demonstrated a parallel recovery in the expression of these key pro-angiogenic factors (Figures 5H,I).

Collectively, these in vivo data provided compelling evidence that endothelial BACH1 is a critical driver of both elevated blood pressure and impaired angiogenesis in hypertension, acting by suppressing a core set of pro-angiogenic factors.

In summary, our study identifies the transcription factor BACH1 as a pivotal molecule in the pathogenesis of hypertension, demonstrating its close association with both elevated blood pressure and impaired angiogenesis. Mechanistically, we reveal that under hypertensive conditions, BACH1 functions as a direct transcriptional repressor of a cohort of key pro-angiogenic factors, including FGF1, VEGFA, ANGPT1 and AGGF1. The coordinate suppression of these factors culminates in the deficient angiogenic response in hypertension (Figure 6). This work not only provides new insights into the molecular basis of hypertensive vascular disorders but also highlights that targeting BACH1 is a promising therapeutic strategy for improving angiogenesis and treating hypertension.