Hypoxia-induced histone lactylation promotes pulmonary arterial smooth muscle cells proliferation in pulmonary hypertension

Abstract

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). Glycolysis plays a crucial role in PH pathogenesis, but the epigenetic mechanisms linking glycolysis to PASMCs proliferation remain unclear. Histone lactylation, a novel post-translational modification derived from glycolytic lactate, may regulate PASMCs proliferation. Primary rat PASMCs were cultured under hypoxia and treated with sodium L-lactate (NaLa) to assess glycolytic activity and histone lactylation. RNA sequencing, RT-qPCR, and Western blotting identified differentially expressed genes (DEGs), while ChIP-qPCR evaluated histone lactylation enrichment at gene promoters. In vivo, a hypoxia-induced PH rat model was used to examine the effect of glycolysis inhibition using oxamate. Mendelian randomization (MR) analysis assessed the causal relationship between placental growth factor (PGF) and PH. Hypoxia and NaLa treatment significantly increased glycolytic activity, lactate production, and histone lactylation, promoting PASMCs proliferation. Transcriptomic analysis identified 157 DEGs, with five key genes (Gbe1, Pgf, Mt2A, Ythdf2 and Gys1) upregulated in response to histone lactylation. ChIP-qPCR confirmed H3K18la enrichment at their promoters. Glycolysis inhibition with oxamate effectively reduced histone lactylation, PASMCs proliferation, and vascular remodeling in hypoxic PH rats. MR analysis identified PGF as a causal factor contributing to PH risk, suggesting a potential therapeutic target. This study reveals that glycolysis-induced histone lactylation drives PASMCs proliferation and vascular remodeling in PH. Targeting lactate metabolism and histone lactylation may provide a novel therapeutic approach.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11010-025-05342-8.

Introduction

Pulmonary hypertension (PH), defined as a mean pulmonary arterial pressure (mPAP) exceeding 25 mmHg (1 mmHg = 0.133 kPa) at rest, is characterized by sustained and progressive elevation of pulmonary vascular resistance, which may ultimately result in right ventricular failure and death. Lung disease and/or hypoxia-induced PH falls under the third category of clinical PH’s five subtypes [1]. Obstructive remodeling of pulmonary arterioles is a remarkable clinical modification associated with hypoxic pulmonary hypertension (HPH), which is mostly evidenced by the hypoxia-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) [2]. Emerging evidence has demonstrated that aberrant glycolysis significantly contributes to PASMCs proliferation during vascular remodeling in PH [3–5]. Nevertheless, the precise mechanisms underlying glycolysis regulation in PASMCs remain incompletely understood.

Lactate, a major end-product of glycolysis, has emerged as an epigenetic regulator through a newly discovered histone post-translational modification—histone lysine lactylation [6, 7]. This modification has been implicated in macrophage polarization and tumor progression, but its role in vascular remodeling, particularly in the context of PH, has not been thoroughly investigated. Moreover, whether glycolysis-derived lactate can promote PASMCs proliferation via histone lactylation remains an open question.

To address this gap, our study investigates the relationship between glycolysis and histone lactylation in hypoxia-induced PASMCs. We aim to uncover whether histone lactylation mediates gene expression changes that contribute to pulmonary vascular remodeling, thereby providing new mechanistic insights into the metabolic—epigenetic regulation of PH and offering potential therapeutic targets.

Methods

Primary rat PASMCs isolation and culture

Primary rat PASMCs are isolated from the pulmonary arteries of five male Sprague–Dawley rats and culture in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) [8]. Cells are passaged using 0.25% trypsin and maintain in a 5% CO₂ atmosphere at 37 °C. Once reaching 80–90% confluence, cells are passaged and seed into six-well plates at ~ 15 × 10⁴ cells/well. After 8 h of starvation, cells are exposed to various treatments, including hypoxia (CoCl₂, 100 µM), NaLa (sodium L-lactate, 20 mM), or oxamate (50 µM), followed by collection for subsequent analysis.

Western blot analysis

Total proteins from cells are extracted using RIPA lysis buffer supplemented with 1 mM PMSF and 1% protease inhibitor cocktail. Protein samples are separated by 10% SDS-PAGE and transfer to PVDF membranes, which are then blocked with 5% non-fat milk for 1 h at room temperature. Membranes are probed overnight at 4 °C with primary antibodies: anti-glucan (1,4-alpha-), branching enzyme 1 (GBE1, 1:500, 20,313-1-AP, Proteintech, USA), anti-placental growth factor (PGF, 1:500, Santa Cruz, USA), anti-metallothionein 2 A (MT2A, 1:500, PA5-120,694, Invitrogen, USA), anti-YTH domain family, member 2 (YTHDF2, 1:500, HA500351, HUABIO, China), anti-glycogen synthase 1 (GYS1, 1:500, ET1611-59, HUABIO, China), and anti-β-actin (1:2000, GB12045-100, Servicebio, China). After incubation with anti-mouse or anti-rabbit IgG (H + L, 1:6000, GB23301/GB23303, Servicebio, China), protein signals are visualized using an ECL reagent kit (Meilunbio, China), and band intensities are quantified using ImageJ software. Protein expression levels are normalized to β-actin [9].

Determination of glycolysis pathway

Glycolysis is assessed by measuring glucose consumption, lactate levels, lactate dehydrogenase (LDH) activity, and cellular pH. Glucose consumption and lactate levels are determined using the Glucose Assay Kit (ab65333, Abcam, USA) and L-lactate Assay Kit (ab65330, Abcam, USA), respectively. LDH activity is measured using the LDH Activity Assay Kit (ab65393, Abcam, USA), and intracellular pH is evaluated using the Fluorimetric Intracellular pH Assay Kit (ab228552, Abcam, USA), following the manufacturer’s instructions.

Cell proliferation assays

Cell proliferation are detected by 5-ethynyl-2’-deoxyuridine (EdU) assay kit (C0075S, Beyotime, China), immunocytochemistry with Ki67 antibody (1:300, GB111499-100, Servicebio, China), and flow cytometry kit (C1052, Beyotime, China), following the manufacturer’s instructions [8].

RNA sequencing (RNA-seq) and bioinformatic analysis

Total mRNA is isolated from cultured PASMCs using a total RNA isolation kit (RC101-01, Vazyme, China), followed by RNA-seq. Differential gene expression is analyzed using the Limma package in R (version 3.6.3) with a cutoff of adjusted P value (Benjamini–Hochberg method) < 0.05 and log2-absolute fold change (FC) > 1. A volcano plot is generated using ggplot2 (version 3.3.3). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs are performed using clusterProfiler (version 3.14.3) and visualized with ggplot2 (version 3.3.3). GO terms are categorized into molecular function (MF), cellular component (CC), and biological process (BP). Gene Set Enrichment Analysis (GSEA) is used to evaluate gene sets, with criteria of normalized enrichment score (|NES|) ≥ 1 and false discovery rate (FDR) ≤ 0.25 [10].

A nomogram is developed based on Venn diagram results to predict the PH prevalence. Calibration curves assess the nomogram’s discrimination capacity, while receiver operating characteristic (ROC) analysis is used to evaluate the model’s performance.

Real-time quantitative polymerase chain reaction analysis (RT-qPCR)

Total mRNA is isolated from cultured PASMCs using cell total RNA isolation kit (RC101-01, Vazyme, China). First-strand cDNA is synthesized and subsequently amplified using Reverse Transcriptase cDNA kit (HRF0032, Heruibio, China). SYBR-Green is used as a fluorophore and qPCR is performed in triplicate using Roche LightCycler96 system (Roche, Switzerland) [11]. The primers used in this study are provided in Supplementary Table 1.

Chromatin immunoprecipitation-quantitative real time PCR (ChIP-qPCR)

Samples are cross-linked with 1% formaldehyde at room temperature, quenching the reaction with glycine. After centrifugation, the cells are washed, lysed, sonicated with working lysis and ChIP buffer provided in the kit. Cross-linked DNA was immunoprecipitated using a ChIP-grade antibody against H3K18la (PTM-1427RM, PTMBIO, China) or IgG as a nonspecific control. The complexes were pulled down with protein A/G beads, reverse cross-linked, and purified for qPCR analysis. Supplementary Table 2 lists the ChIP-qPCR primers used in this study, which are designed using Primer-BLAST (NCBI).

PH models

A total of 24 male Sprague–Dawley (SD) rats (150-180 g, Shanghai SLACCAS Laboratory Animal Co. Ltd, China) are randomly divided into three groups, including control (Ctrl), Su/Hx-PH (Su/Hx-PH) and Su/Hx-PH + oxamate (Su/Hx-PH + oxa), with eight rats per group. The Ctrl group is maintained under normoxic conditions, while the remaining 16 rats receive a single subcutaneous injection of Su5416 (20 mg/kg) and are exposed to chronic hypoxia (10% O₂) for 3 weeks, followed by 2 weeks of normoxia to establish the PH model. Among them, eight rats receive daily intraperitoneal injections of the LDH inhibitor oxamate (750 mg/kg), while the remaining rats are administered an equal volume of saline. All animal procedures adhere strictly to the “Guidelines for the Care and Use of Laboratory Animals” (National Academy of Sciences, 2011) and are approved by the Laboratory Animal Welfare and Ethics Committee of Fujian Medical University (Approval No. 2017-070, Fuzhou, China).

Hemodynamic analysis and morphometric analysis

Hemodynamic measurements are performed using a previously established protocol [12], in which rats are anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). Right ventricular pressure (RVP) is measured by inserting a polyethylene catheter, connected to a pressure transducer, into the right ventricle through the right external jugular vein. The catheter is then advanced into the pulmonary artery to monitor pulmonary arterial pressure (PAP). Following the procedure, the hearts and lungs are excised by thoracotomy. mPAP and right ventricular hypertrophy index (RVHI, calculated as RV/(LV + S)) are assessed [13].

Lung tissues are preserved in 4% paraformaldehyde, embedded in paraffin, and section into 5-μm-thick slices. These sections are stained with Hematoxylin&Eosin (H&E) for histologic and collagen analysis. The sections are examined under a Nikon 80i light microscope (Nikon, Japan). Pulmonary vascular remodeling is evaluated by measuring H&E-stained lung sections [14]. Small pulmonary arteries (external diameter 50–100 µm) are randomly selected, and vessel wall thickness and vascular area are quantified using ImageJ software. Wall thickness percentage (WT%) is calculated as (external diameter − internal diameter)/external diameter × 100%, and wall area percentage (WA%) is calculated as (total vascular area − luminal area)/total vascular area × 100%.

After deparaffinization and antigen retrieval, lung tissue sections are double immunostained for α-smooth muscle actin (α-SMA) and co-differentially expressed genes (co-DEGs) [15]. Fluorescent images of α-SMA (green, 1:500, GB111364-100, Servicebio, China), 4′,6-diamidino-2-phenylindole (DAPI) (blue, 1:1000, G1012-10ML, Servicebio, China), and co-DEGs (GBE1, red, 1:500, 20,313-1-AP, Proteintech, USA; PGF, red, 1:500, Santa Cruz, USA; MT2A, red, 1:500, PA5-120,694, Invitrogen, USA; YTHDF2, red, 1:500, HA500351, HUABIO, China; GYS1, red, 1:500, ET1611-59, HUABIO, China) are captured using a Nikon 80i microscope (Nikon, Japan).

Two samples Mendelian randomization (TSMR)

A TSMR analysis is conducted using summary-level data from the IEU Open GWAS database (https://gwas.mrcieu.ac.uk/datasets). The exposure data are obtained from the PGF GWAS summary dataset (GWAS ID: prot-b-66), and the outcome data are derived from the PH GWAS summary dataset (GWAS ID: finn-b-I9_HYPTENSPUL). The demographics of the patients included in the GWAS summary datasets are presented in Supplementary Table 3. For PGF exposure, single nucleotide polymorphisms (SNPs) at the genome-wide significance threshold (P < 5 × 10⁻⁸) are selected, and linkage disequilibrium (LD) is estimated between SNPs to identify independent genetic variants (r² < 0.01). The causal effect of PGF levels on PH is estimated using several Mendelian randomization methods: MR-Egger regression, weighted median, inverse-variance weighted (IVW), simple mode, and weighted mode. The TSMR analysis is carried out using the “Two Sample MR” package in R (version 3.6.3). The threshold for statistical significance for evidence of pleiotropy is set at P < 0.05.

Statistical analysis

Results are presented as the mean ± standard deviation (SD). Comparisons among multiple groups are made using a one-way analysis of variance (ANOVA), followed by the Least Significant Difference (LSD) test. A P value of < 0.05 is considered statistically significant. All experiments are performed in triplicate, unless otherwise stated. All these animal procedures are strictly in accordance with the recommendations in the “Guidelines for the Care and Use of Laboratory Animals (National Academy of Sciences, 2011)”. This study is approved by the Laboratory Animal Welfare and Ethics Committee of Fujian Medical University (Approval No. 2017-070, Fuzhou, China).

Results

Histone lactylation induced by lactate accumulation drove PASMCs proliferation

As shown in Fig. 1A–D, hypoxia and NaLa treatment led to increased glucose consumption, elevated lactate levels, enhanced LDH activity, and a more acidic cellular pH. In addition, both hypoxia and NaLa induced higher expression of L-lactyl lysine (Pan-Kla) and lactylation of histone H3 at lysine 18 (H3K18la), which were reversed by the glycolytic inhibitor oxamate (Fig. 1E and F). Furthermore, oxamate mitigated the proliferative effects of hypoxia and NaLa on PASMCs (Fig. 1G–K).

Fig. 1.

Histone lactylation induced by lactate accumulation drives PASMCs proliferation. A Relative glucose consumption levels in PASMCs. B Relative lactate levels in PASMCs. C Relative LDH activity in PASMCs. D Cellular PH value in PASMCs. E and F Representative Western blot showing global lactylation and H3K18la levels in PASMCs under normoxia and hypoxia, with or without oxamate (50 μM) incubation. G and H Representative EdU (red) images, with quantification of the percentage of EdU-positive nuclei. Scale bar = 150 μm. I Representative immunofluorescence images of PASMCs stained for Ki67. Scale bar = 150 μm. J and K Representative flow cytometry images, with quantification of cell cycle distribution. Data represent mean ± SEM; *P < 0.05 vs. Ctrl; #P < 0.05 vs. hypoxia or NaLa; two-way ANOVA and Bonferroni’s post-test

Screening and identification of co-DEGs in PH induced by hypoxia, lactate and MCT

The volcano plot in Fig. 2A illustrates the DEGs in response to hypoxia and NaLa treatment. A Venn diagram was used to identify overlapping DEGs between the hypoxia-upregulated and NaLa-upregulated gene sets, revealing 157 DEGs (Fig. 2B). GO analysis of these DEGs indicated enrichment in the BP term “pyruvate metabolic process” (Fig. 2C), suggesting a critical role of glycometabolism in PH. In terms of MF, the DEGs were enriched in “carbohydrate kinase activity”, “carbohydrate binding”, and “glucose binding”, highlighting their involvement in PASMCs glycolytic processes. In addition, KEGG pathways included “Glycolysis/Gluconeogenesis”, “HIF-1 signaling pathway”, “Carbon metabolism”, and “Galactose metabolism”, further implicating glycolytic and metabolic pathways in PH. GSEA revealed that these DEGs were also enriched in pathways related to “Cell Cycle”, “DNA Replication”, “Oxidative Phosphorylation”, and “Cytokine Receptor Interaction” (Fig. 2D), hinting that hypoxia- and lactate-related high expression genes were associated with proliferation and mitochondrial metabolism.

Fig. 2.

Histone lactylation transcriptionally activated Gbe1, Pgf, Mt2A, Ythdf2 and Gys1 in PASMCs. A Volcano plot showing DEGs in PASMCs under hypoxia or NaLa treatment compared to Ctrl. B Venn diagram depicting upregulated genes common to hypoxia and NaLa conditions. C and D The 157 genes highlighted in red in the Venn diagram were subjected to GO enrichment, KEGG pathway, and GSEA analysis. E and F Venn diagram showing the intersection of the 157 upregulated genes and 465 upregulated genes identified in response to MCT administration, revealing five co-DEGs. G Nomogram illustrating the five co-DEGs to assess PH risk. H and I Calibration curve and ROC curve evaluating the predictive performance of the PH risk model. J Relative mRNA expressions of Gbe1, Pgf, Mt2A, Ythdf2 and Gys1 under normoxia and hypoxia, with or without oxamate (50 μM) incubation. K and L Representative Western blot showing the protein expression levels of GBE1, PGF, MT2A, YTHDF2 and GYS1 under normoxia and hypoxia, with or without oxamate (50 μM) incubation. M ChIP-qPCR analysis showing H3K18la enrichment at the Gbe1, Pgf, Mt2A, Ythdf2 and Gys1 promoters in PASMCs under normoxia and hypoxia, with or without oxamate (50 μM) incubation. Data represent mean ± SEM; *P < 0.05 vs. Ctrl; #P < 0.05 vs. hypoxia; two-way ANOVA and Bonferroni’s post-test

To identify shared genes between DEGs upregulated in hypoxia and NaLa treatment, and those upregulated in MCT-induced PH, five co-DEGs were identified (Gbe1, Pgf, Mt2A, Ythdf2 and Gys1, Fig. 2E and F). A nomogram was constructed for predicting PH based on these co-DEGs (Fig. 2G). The calibration curve of the nomogram demonstrated a good agreement between actual observation and prediction (Fig. 2H) and the ROC curve was displayed in Fig. 2I (AUC: 0.930, 95% CI 0.896–0.965).

According to the results of RT-qPCR, marked elevations of Gbe1, Pgf, Mt2A, Ythdf2 and Gys1 in PASMCs under hypoxic exposure were confirmed, while significant reductions of those were found with the oxamate administration (Fig. 2J), consistent with the results observed by Western blot (Fig. 2K and L).

Hypoxia promotes co-DEGs expression by linkage between H3K18la and candidate gene promoters

To investigate a potential epigenetic mechanism underlying the hypoxia-induced upregulation of co-DEGs, ChIP-PCR analysis was performed to assess the enrichment of H3K18la in the promoter regions of these genes. After ChIP using an antibody specific to H3K18la, followed by qPCR for the co-DEGs, it was found that hypoxia significantly promoted both the expression and H3K18la enrichment at the promoter regions of Gbe1, Pgf, Mt2A, Ythdf2 and Gys1 (P < 0.05, Fig. 2M).

Glycolytic inhibitor attenuates hypoxia-induced pulmonary vascular remodeling in hypoxic PH rats

As outlined in the experimental workflow (Fig. 3A), rats were randomly assigned to Ctrl, Su/Hx-PH, and Su/Hx-PH + oxa groups to evaluate the therapeutic potential of oxamate in pulmonary hypertension. In vivo, oxamate attenuated hypoxia-induced elevation of mPAP, RVP and RVHI (Fig. 3B and D). In addition, oxamate mitigated pulmonary artery medial thickness in PH, as evidenced by a decrease in WA% and WT% (Fig. 3C and D). Furthermore, protein levels of co-DEGs were assessed in isolated pulmonary arterioles, revealing that the expression of co-DEGs in hypoxic pulmonary arterioles was significantly increased, an effect that was mitigated by oxamate treatment (Fig. 3E–G).

Fig. 3.

Glycolytic inhibitor attenuated hypoxia-induced pulmonary vascular remodeling in hypoxic PH rats. A Schematic diagram of the in vivo experimental design. B Representative traces of RVP and mPAP. C Representative H&E images of lung sections. Scale bar = 100 μm. D Quantification of the mPAP, RVP, RVHI, WA% and WT%. E Representative immunofluorescence images of lung sections stained for α-SMA (red), DAPI (blue) and co-DEG genes (green). Scale bar = 50 μm. F and G Representative Western blot showing the protein expression levels of GBE1, PGF, MT2A, YTHDF2 and GYS1 in Su/Hx-PH with or without oxamate (750 mg/kg) administration. Data represent mean ± SEM; *P < 0.05 vs. Ctrl Group; #P < 0.05 vs. Su/Hx-PH Group; two-way ANOVA and Bonferroni’s post-test

Verification of the causal relationship between PGF and PH based on TSMR

According to the nomogram results, PGF was determined as the risk factor with the highest contribution, prompting further exploration of a potential causal relationship between PGF and and PH. PGF was considered the exposure factor, and PH was the outcome variable. A total of five SNPs were selected as IVs, with F values greater than ten. The intercept was close to zero (Cochran’s Q test P = 0.742; Egger intercept-derived P = 0.883), indicating that no horizontal pleiotropy of the IVs, suggesting that they were unlikely to influence the results of TSMR analysis (Fig. 4A and C). In the absence of horizontal pleiotropy of IVs, PGF (IVW result: OR 1.266, 95% CI 1.057–1.963, P = 0.019) was identified as causal factor contributing to the increased risk of PH (Fig. 4A and B). Subsequently, the sensitivity analysis used the leave-one-out method to remove SNPs one by one, and the causal effects of the remaining SNPs were compared with the TSMR analysis results of all SNPs to determine whether the causal association was driven by a single IV, indicating that the TSMR analysis results were robust and not influenced by any single IV (Fig. 4D and E).

Fig. 4.

PGF was a causal factor contributing to increased PH risk. A Mendelian randomization analysis demonstrating a causal association between PGF and PH risk. B Forest plot illustrating the effect estimates of PGF on PH. C Scatter plot showing the relationship between PGF levels and PH risk. D Sensitivity analysis assessing the robustness of the causal effect of PGF on PH. E Funnel plot evaluating potential biases in the Mendelian randomization analysis of PGF and PH risk

Discussion

In this study, the potential role of histone lactylation in hypoxia-induced PH was investigated. Elevated levels of glycolysis, lactate, and histone lactylation were observed in hypoxic PASMCs. A total of 157 DEGs were found to be upregulated by hypoxia or lactate treatment, primarily involving glycolysis and HIF-1 signaling pathways. Notably, five genes (Gbe1, Pgf, Mt2a, Ythdf2, and Gys1) from these 157 DEGs were also detected in lung tissues from MCT-induced PH. Glycolysis inhibition was shown to reverse hypoxic pulmonary vascular remodeling and the expression of co-DEGs in hypoxic PH, likely due to a reduction in lactate production, which, in turn, diminished the interaction between H3K18la and the promoters of these candidate genes. Furthermore, PGF was identified as a causal factor contributing to the increased risk of PH.

Under hypoxic conditions, most eukaryotic cells increase glycolysis to maintain ATP levels, rather than relying predominantly on mitochondrial respiration. This metabolic switch is regulated by several pathways at the transcriptional level, including hypoxia-inducible factor-1α (HIF-1α), which promotes the upregulation of glycolytic enzymes [16]. While this increase in glycolytic flux helps maintain bioenergetic homeostasis during hypoxia, its role in supporting cell survival and growth, particularly in proliferative cells, has been extensively studied [17]. Lactate, rather than merely a byproduct of fermentative glycolysis, acts as a crucial signaling molecule. It plays an essential role in sustaining angiogenesis, evading immune surveillance, and reprogramming energy metabolism. Recent studies have established lactate production in hypoxic microenvironments as a key facilitator of angiogenesis [18, 19]. Several studies suggested that lactate-driven H3K9 lactylation in endothelial cells (ECs) modulates histone deacetylase 2 (HDAC2) expression through a feedback loop, thereby regulating VEGF-induced angiogenesis [20]. In addition, lactate enhanced the production of vascular endothelial growth factor (VEGF) and its receptor VEGFR2 production in ECs [21]. Consistent with our findings, hypoxic PASMCs oxidized glucose anaerobically, and the resultant lactate facilitated PASMCs proliferation, which could be reversed by an LDH inhibitor.

Epigenetics, an emerging field in cancer therapy, also has potential for PH, given the growing evidence that epigenetic regulation plays a crucial role in PH [22]. Recent researches have shown that lactate functions as an epigenetic modifier, modulating gene expression via histone lactylation, a newly discovered form of histone modification [6, 23]. In this study, we demonstrated that lactylation of lysine residues on histones was promoted by increased lactate pool in hypoxia. While more direct manipulation of histone lactylation was not feasible, the use of an LDH inhibitor significantly reduced histone lactylation, PASMCs proliferation, and pulmonary vascular remodeling, which highlighted the potential role of epigenetic modification mediated by a metabolic intermediate of glycolysis in PH. Furthermore, 157 genes upregulated in PASMCs either under hypoxia or upon lactate treatment were primarily enriched in pathways related to the cell cycle and DNA replication, indicating that lactate was important for regulating hypoxia-induced proliferation. Our results revealed a novel mechanism in which hypoxia-induced histone lactylation, driven by elevated lactate production, contributed to the pathophysiological progression of PH.

Glycogen synthase 1 (GYS1), a rate-limiting enzyme in glycogen synthesis, is upregulated by HIF-1α signaling under hypoxic conditions, leading to increased glycogen accumulation in cells [24]. The downstream target of HIF-1α, glycogen branching enzyme 1 (GBE1), has been implicated in various cellular processes, including lung cancer cell proliferation, migration, invasion, angiogenesis, and metastasis under hypoxia [25]. Our study demonstrated that GYS1 and GBE1 expression levels were elevated in hypoxic PASMCs, suggesting an enhanced glycogen synthesis and branching process under hypoxic stress in PASMCs. However, previous studies have reported that both GYS1 and GBE1 are downregulated in pulmonary tissues from patients with PH [26]. This apparent discrepancy may reflect cell type-specific responses or dynamic changes in glycogen metabolism during different stages or microenvironments of disease progression. Further investigation is warranted to elucidate the regulatory mechanisms and functional consequences of altered glycogen metabolism in PH pathogenesis. Placenta growth factor (PGF), a member of VEGF family, is a well-established angiogenic growth factor. PGF is a multitasking cytokine able to stimulate angiogenesis through both direct and indirect mechanisms and is involved in pathologic angiogenesis, such as tumor neovascularization, tissue ischemia-induced vascular regeneration, and wound healing. Elevated PGF mRNA expression has been observed in hypoxic lung tissue, and PGF has been shown to induce endothelin-1 expression, promoting pulmonary vasoconstriction by erythroid cells [26]. Hypoxia-induced angiogenesis in PH is hypothesized to be significantly influenced by PGF, which aligns with our previous findingss that PGF is involved in MCT-induced PH [8]. Metallothionein 2 A (MT2A) regulates key processes such as autophagy, cell proliferation, angiogenesis, and vasoconstriction, which are essential in PH pathophysiology [27]. However, there is limited direct evidence regarding the role of MT2A in PH. In our prior studies, pulmonary vascular remodeling was reversed by suppressing metal-regulatory transcription factor 1 (MTF-1), the upstream transcription factor of MT2A, which was found to be overexpressed in our study [8]. YT521-B homology domain family 2 (YTHDF2), an N⁶-methyladenosine (m⁶A) modification reader, is highly involved in multiple BPs, including migration, proliferation, apoptosis, cell cycle, cell viability, cell adhesion, differentiation, and inflammation [28]. Recent work by Wang et al. [29] established that hypoxia stimulated YTHDF2 expression in PASMCs, which is consistent with our findings. Furthermore, YTHDF2 recognize m6A-modified phosphatase and tensin homolog (PTEN) mRNA, facilitating its degradation and ultimately inducing PASMC migration and proliferation through the PI3K/Akt signaling pathway. In addition, Yu et al. [28] discovered that elevated lactate-induced histone lactylation promoted tumorigenesis by activating YTHDF2. However, to date, there is no direct evidence linking hypoxia-induced upregulation of YTHDF2 expression with histone lactylation in PH.

In addition to the prominent staining of co-DEG signals in the lung parenchyma, our immunofluorescence analysis revealed that these genes were expressed at relatively low levels in vascular smooth muscle cells, suggesting that the upregulation of co-DEGs under hypoxic conditions is not restricted to PASMCs but may also involve other pulmonary cell types. Previous studies have indicated that alveolar epithelial cells, interstitial fibroblasts, and inflammatory cells can actively respond to hypoxic stress, participating in both adaptive and pathologic remodeling of the lung microenvironment [30, 31]. The substantial expression of co-DEGs in these non-vascular compartments reflects the broader impact of hypoxia-induced metabolic and epigenetic alterations, such as histone lactylation, across diverse cell populations. This cellular heterogeneity underscores the complexity of PH pathogenesis and points to the necessity of further research employing cell-type-specific approaches (such as single-cell RNA sequencing or lineage-tracing models) to precisely identify the sources and functions of these gene expression changes. A more comprehensive understanding of how vascular and non-vascular cells interact and contribute to pulmonary vascular remodeling may reveal new targets for therapeutic intervention in PH.

Our findings provided important mechanistic insight into the pathogenesis of PH by establishing a novel link between hypoxia-driven metabolic reprogramming and epigenetic regulation via histone lactylation. By identifying key genes (such as PGF, GBE1, MT2A, YTHDF2, and GYS1) that were regulated through this axis, we revealed how metabolic stress could orchestrate pulmonary vascular remodeling at the epigenetic level. Importantly, these results highlighted the translational potential of targeting glycolytic flux and histone lactylation as therapeutic strategies for PH. Given that metabolic intermediates like lactate directly modulate gene expression and promote pathologic cell proliferation, interventions that inhibit glycolysis or modulate histone modifications may represent promising avenues for clinical management. Several metabolic inhibitors, including LDH inhibitors, have already shown clinical utility in other disease settings [7, 32], underscoring the feasibility of repurposing such agents for PH. Furthermore, our findings suggested that monitoring lactate levels and histone lactylation status might serve as novel biomarkers for disease activity and therapeutic response, thereby facilitating more precise patient stratification and personalized treatment strategies. Collectively, this study not only advanced our understanding of PH pathobiology but also provided a conceptual framework for developing metabolism- and epigenetics-based diagnostic and therapeutic approaches, with significant clinical translational value.

Inevitably, limitations also existed in this study. First, the sequencing results utilized in these analyses were derived from rats rather than humans, a factor that could potentially compromise the clinical applicability of the results. Second, there are few available databases on PASMCs derived from MCT-induced PH, necessitating the use of the GEO database for co-DEG analysis of MCT-induced PH rat lung tissue. Third, this study relied solely on transcriptomic data to indirectly explore the role of hypoxia-induced lactylation-related genes in PH. Future studies utilizing lactylation omics analysis will be required to identify the specific lactation sites on proteins under hypoxic conditions.

Conclusions

In summary, this study proposed a new paradigm for regulating the proliferation-related genes expression in PH. As illustrated in Fig. 5, hypoxia stimulated the lactate production, and lactate, functioning as an epigenetic regulator, enhanced the expression of Gbe1, Pgf, Mt2A, Ythdf2 and Gys1 via histone lactylation, which in turn promoted hypoxic pulmonary arterial remodeling. By elucidating a novel downstream effector of increased glycolysis in PH onset, these findings provide proof of concept for histone lactylation-based anti-remodeling therapies.

Fig. 5.

Schematics diagram depicting the key findings of this study. Hypoxia-induced upregulation of histone lactylation promoted PASMCs proliferation and pulmonary vascular remodeling by transcriptionally enhancing the expression of GBE1, PGF, MT2A, YTHDF2, and GYS1

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Ai Chen and Zhihai Chen conceived and designed the study. Ai Chen, Zhihai Chen, Bangbang Huang and Guili Lian performed data curation. Ai Chen, Zhihai Chen conducted data analysis and drafted the initial manuscript. Li Luo and Liangdi Xie made a critical revision to the manuscript for important intellectual content. All authors read the manuscript and approved the final draft.

Funding

This study was supported by Scientific research funding for the introduction of talents by the First Affiliated Hospital of Fujian Medical University [YJRC4183], National Natural Science Foundation of China under Grant [82370351] and Joint Funds for the Innovation of Science and Technology, Fujian Province [2020Y9108].

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Fujian Medical University (Approval No. 2017-070, Fuzhou, China).

Consent to participate

Informed consent was obtained from all individual participants included in the study.