Authors: Tiantian Mu, Boshuo Guo, Chuqi Xiang, Ziwen Liu, Jing Ren, Manling Liu, Pengtao Zhao
Categories: Review, Pulmonary hypertension, Hypoxic microenvironment, Vascular remodeling, Immunoinflammation
Source: Respiratory Research
Authors: Tiantian Mu, Boshuo Guo, Chuqi Xiang, Ziwen Liu, Jing Ren, Manling Liu, Pengtao Zhao
Pulmonary hypertension (PH) is a serious pulmonary vascular disease characterized by a progressive increase in pulmonary vascular resistance and abnormally high pulmonary arterial pressure. The hypoxic microenvironment plays an important role in its development. Studies have indicated that early exposure of pulmonary vasculature to hypoxia in the hypoxic microenvironment triggers an adaptive response in the organisms and has a homeostatic regulatory effect. However, under prolonged hypoxic stimulation, pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs) can be induced to abnormally proliferate and migrate through endothelial cell dysfunction and endothelial-mesenchymal transition. This leads to irreversible pulmonary vascular remodeling, which ultimately results in PH formation. Core components of the hypoxic microenvironment include hypoxia-inducible factors (HIFs) through a complex regulatory network, metabolic reprogramming in the microenvironment (glucose metabolism, lipid metabolism, and amino acid metabolism), an overabundance of reactive oxygen species and redox imbalance, reprogramming of the immuno-inflammatory microenvironment, regulation of cell death patterns (apoptosis resistance, iron death, and autophagy imbalance), mechanical stress and cytoskeletal dynamics, non-coding RNA regulatory networks (miRNA, IncRNA, and circRNA), microbial-host interactions (gut flora metabolites), epigenetic regulation (DNA methylation, histone modification, and RNA modification) and Transient Receptor Potential (TRP) Channels and Calcium Signaling Regulation. These processes are interconnected in the organisms to induce or promote aberrant proliferation and migration of PASMCs and PAECs, which are the pathogenic mechanisms resulting in PH.
Current clinical treatments for PH include endothelin receptor antagonists, drugs targeting cyclic guanosine monophosphate production, phosphodiesterase-5 inhibitors, and prostacyclin analogs. However, novel targeted drugs against HIF-1ɑ remain under development. Oxygen therapy and mechanical ventilation, gene therapy, and molecularly targeted interventions (modulation of the RhoA/ROCK pathway or non-coding RNAs) can improve hypoxemia. Future studies must integrate multi-omics data, incorporate artificial intelligence to accelerate drug development, and focus on gender and individualization to achieve precision therapy. In conclusion, an in-depth analysis of the mechanism of the hypoxic microenvironment in PH will provide the fundamental basis for developing more effective therapeutic strategies.
Pulmonary hypertension (PH) is a serious pulmonary vascular disease characterized by a progressive increase in pulmonary vascular resistance and an abnormal increase in pulmonary artery pressure [1]. The primary pathological changes are pulmonary vascular remodeling (PVR) and pulmonary vasoconstriction, which raise pulmonary circulatory pressure, increase right ventricle load, and ultimately lead to right heart failure or even death [2–4]. Under normal physiological conditions, the human body exchanges gases through the lungs, and sufficient oxygen is an important condition for maintaining the normal physiological function of an organism. When the body is in low oxygen, the adaptive response can be triggered early on to perform homeostatic regulation to redistribute blood flow in the pulmonary vasculature and maintain the body’s normal ventilation/blood flow ratio. Multiple studies have suggested that when pulmonary vasculature is subjected to prolonged hypoxic stimulation, components of the hypoxic microenvironment interact with one another to upregulate vasoconstrictor factors and inhibit vasodilator substances through the activation of hypoxia-inducible factor (HIF) signaling pathway [5, 6]. This results in sustained constriction of the pulmonary vasculature, inducing proliferation of pulmonary vascular smooth muscle cells (PASMCs), activation of fibroblasts, and infiltration of inflammatory cells, irreversible PVR, ultimately elevating pulmonary artery pressure and exacerbating right heart failure. Additionally, hypoxia-induced metabolic reprogramming and oxidative stress can further promote the pathological process of PH. Consequently, a hypoxic microenvironment is a key mechanism in PH development.
PH is a severe chronic pulmonary vascular disease characterized by a mean pulmonary artery pressure (mPAP) ≥ 20 mmHg measured by right heart catheterization at rest. As the disease progresses, patients can experience symptoms including dyspnea, fatigue, and chest pain. Right heart failure frequently results in death during the advanced stages of the disease [7]. PH is clinically harmful and has a significant impact on the quality of life and prognosis of patients.
According to the 2022 European Heart Association/European Respiratory Society Guidelines for PH diagnosis and treatment, it can be classified into the following (1) arterial PH, including idiopathic PH and drug- and toxin-induced PH; (2) PH associated with left heart disease, including valvular heart disease and heart failure with preserved or reduced ejection fraction; (3) PH associated with lung disease or hypoxia, including obstructive lung disease or emphysema, restrictive lung disease, and hypoventilation syndrome; (4) PH associated with pulmonary artery obstruction and chronic thromboembolic PH; (5) PH with unknown or multifactorial mechanisms, hematological disorders, systemic diseases, and metabolic disorders [8].
PVR is the central pathological change in PH. It is manifested by remodeling of the three vessel wall layers of small pulmonary arteries, apoptosis and proliferation of intimal endothelial cells, proliferation of smooth muscle cells in the middle membrane, fibrosis of the outer membrane, and infiltration of inflammatory cells and dendritic cells (Fig. 1) [9, 10]. These processes result in thickening of the vessel wall, narrowing of the lumen, and increased pulmonary vascular resistance. Smooth muscle cell proliferation is an important part of PVR. Endothelial dysfunction, including endothelial cell injury, decreased nitric oxide (NO) synthesis, and increased release of vasoconstrictor factors, disrupts the balance between vasodilation and constriction [7]. This stimulates smooth muscle cells to proliferate abnormally and migrate to the intima, thus promoting PVR process. Furthermore, during mesenchymal transition, epithelial or endothelial cells are transformed into mesenchymal cells, acquiring the ability to migrate and proliferate, further exacerbating PVR [11].
Fig. 1Pathological changes in PH. Massive endothelial cell apoptosis and proliferation, mesangial smooth muscle cell proliferation, epicardial fibrosis, and infiltration of inflammatory cells and dendritic cells cause hemopulmonary tubular remodeling, resulting in thickening of the vessel wall and narrowing of the lumen. Created by Figdraw
When the body is stimulated by hypoxia, it triggers oxidative stress, immuno-inflammation, dysregulation of vasoactive molecules, and intercellular interactions to form a complex pathological network, forming a hypoxic microenvironment. The core components of this hypoxic microenvironment include a transcriptional regulatory network activated by HIFs, metabolic reprogramming (enhanced glycolysis, lipid accumulation, and abnormalities in amino acid metabolism), an imbalance of oxidative stress (accumulation of reactive oxygen species (ROS) from mitochondrial and NADPH oxidase-derived ROS accumulation accompanied by a breakdown of antioxidant system), immune-inflammatory dysregulation (macrophage polarization imbalance, neutrophil extracellular trap formation, and T-cell depletion), aberrant cell death patterns (smooth muscle cell apoptosis resistance and endothelial cell autophagy imbalance), altered mechanical stress and cytoskeletal dynamics (activation of the Yes-associated protein (YAP)/transcriptional co-activator with a PDZ-binding motif (TAZ) and RhoA/ROCK pathways), disruption of non-coding RNA regulatory network disorders (miRNA, lncRNA, and circRNA-mediated gene silencing or pro-proliferative signaling), microbial-host interactions (exacerbation of vasoconstriction and inflammation by gut flora metabolites through the gut-pulmonary axis), epigenetic modifications (reprogramming of gene expression driven by DNA methylation, histone modification, and RNA methylation) and transient receptor potential (TRP) channels and calcium signaling regulation.
The hypoxic microenvironment plays a dual role in PH development. Under physiological conditions, pulmonary vasculature initially constricts in response to hypoxic stimuli, triggering adaptive responses to maintain optimal arterial oxygenation. These responses include increased erythropoietin secretion, which enhances oxygen-carrying capacity to maintain tissue oxygen supply. However, under persistent or severe hypoxic conditions, various components of the hypoxic microenvironment form a vicious circle through multidimensional cross-talk, including HIFs-metabolic-oxidative stress axis and immune-mechanical stress linkage, which together drive the abnormal proliferation and migration of pulmonary artery endothelial cells (PAECs) and PASMCs. This results in irreversible PVR and progression of PH[6, 12].
The core components of the hypoxic microenvironment, as a key causative factor in PH development, can be summarized as a “three-dimensional imbalance”: a vicious cycle of disturbed polarization of cellular function, amplification of molecular signaling cascades, and metabolic reprogramming. First, endothelial cell dysfunction, aberrant proliferation of PASMCs, and inflammatory polarization of immune cells induce PVR [13]. Second, HIF directly promotes PASMCs’ proliferation and endothelial dysfunction by modulating glycolysis, inflammatory factor expression, apoptosis resistance, and vascular contraction and relaxation [14, 15]. ROS accumulation results in an oxidative-antioxidant imbalance that activates pro-inflammatory signaling pathways. This results in a positive feedback effect of “oxidative damage-inflammation amplification-vascular remodeling”, promoting PH development [16]. In addition, the hypoxic microenvironment promotes microenvironmental acidification through abnormal glucose metabolism (Warburg effect), lipid accumulation, and amino acid metabolism imbalance, providing an energy base for abnormal cell proliferation [17, 18].
Importantly, these mechanisms do not operate in isolation but form an interactive synergistic network that collectively drives the pathogenesis and progression of PH. For instance, HIF not only directly regulates downstream gene expression but also achieves transcriptional amplification and sustained activation through epigenetic mechanisms (DNA methylation, histone modifications [19]. Metabolites derived from metabolic reprogramming (lactate, lipotoxic molecules) can further modulate oxidative stress and immune-inflammatory responses. Oxidative stress potentiates hypoxic signaling by modifying HIF and the activity of epigenetic enzymes [20, 21]. Moreover, activation of the YAP/TAZ signaling pathway and mechanical stress in a hypoxic microenvironment synergistically interact with each other, as well as microbial gut flora, through metabolite regulation through the “gut-lung axis” to drive the pathological process of PH[22, 23].
HIFs have important roles in regulating and adapting to a hypoxic environment and are core transcription factors in the hypoxic microenvironment. HIF has three ɑ subunits and two β subunits. HIF-ɑ and HIF-β form a heterodimer in the nucleus and induce the expression of multiple genes. HIF-ɑ is primarily consist of isoforms, including HIF-1ɑ, HIF-2ɑ, and HIF-3ɑ[24]. They play different cell-specific roles in PH development and are interconnected with hypoxic microenvironment-induced cellular dysfunction, metabolic reprogramming, and immune-inflammatory responses, which contribute to the pathological process of PH. HIF-1ɑ and HIF-2ɑ have been extensively studied, and these two subunits have overlapping roles and functions.
Under normoxic conditions, HIF-ɑ is degraded by the ubiquitin-proteasome system through hydroxylation mediated by prolyl hydroxylases (PHDs) and factor inhibiting HIF (FIH) [25]. However, under hypoxic conditions, the activity of PHDs and FIH decreases due to the impairment of oxygen-dependent hydroxylation reactions [26]. This prevents HIF-ɑ subunits from being degraded, leading to their stable accumulation. This process is the core step in HIF activation.
HIF-1ɑ is extensively expressed in different cells, and its expression is significantly induced under hypoxic conditions. In the rat hypoxic PH model, it was shown that HIF-1ɑ inhibitors can reduce the severity of PH. This indicates that HIF-1ɑ plays an important role in the progression of PH. In PASMCs, HIF-1ɑ promotes cell proliferation and migration by regulating the expression of a series of target genes. First, it upregulates the expression of vascular endothelial growth factor (VEGF), increasing vascular permeability and neovascularization while activating the platelet-derived growth factor (PDGF) signaling pathway [27]. This promotes the proliferation and migration of PASMCs and increases PVR [28]. Second, in lung endothelial cells, HIF-1ɑ regulates the expression of NO synthase (eNOS) and affects vasodilatory function. HIF-1ɑ overactivation significantly reduces eNOS expression and NO production, which in turn increases vasoconstriction and promotes PH development. HIF-1ɑ accumulation increases oxidative stress and the inflammatory microenvironment by promoting the expression of pro-inflammatory mediators (IL-6) and cell adhesion molecules (ICAM-1), a pathological state that further inhibits the activity of eNOS and its stability [29, 30]. Furthermore, HIF1ɑ promotes cell survival and proliferation by regulating cellular metabolic pathways, including increasing glycolysis. This mechanism is closely associated with the metabolic reprogramming induced by the hypoxic microenvironment, which will be discussed in detail in the section “Abnormalities of glucose metabolism”.
HIF-1ɑ is the main regulatory factor of the early hypoxic response, while long-term hypoxia triggers an activity switch from HIF1ɑ to HIF2ɑ, and this switch is crucial for the progression of PH[31].
HIF-2ɑ is highly expressed in lung endothelial cells and certain immune cells. In lung tissues of patients with PH, upregulated expression of HIF-2ɑ promotes the proliferation and survival of pulmonary endothelial cells. It regulates the inflammatory response, which can further exacerbate the process of PVR [32]. HIF-2ɑ expression is upregulated in macrophages and other immune cells, which promotes the polarization and functional alterations of these cells [33]. In the hypoxic microenvironment of PH, HIF-2ɑ promotes the polarization of M1-type macrophages, increases the release of pro-inflammatory factors, and promotes the inflammatory response, thereby exacerbating the inflammatory and remodeling processes in the pulmonary vasculature. The specific mechanisms of which are explained in detail in the section “Macrophages”. Additionally, HIF-2ɑ enhances Myc activity by promoting the binding of the transcription factor Myc to its co-activator. Myc overexpression increases the expression of pro-apoptotic genes and accelerates cell death. Moreover, HIF-1ɑ is a negative regulator of Myc, which has the opposite effect to HIF-2ɑ[34]. These effects directly influence the apoptotic process and determine cell survival, thus directly affecting PVR and maintenance of PH, which can play a dual role in PH progression.
It was found that both HIF-1ɑ and HIF-2ɑ are upregulated in pulmonary arterial smooth muscle cells and lung tissue lysates; however, inhibition of HIF-2ɑ results in a more significant improvement in pulmonary vascular hemodynamics [35]. HIF-2ɑ specific inhibitors can reduce pulmonary vascular remodeling and the expression of PH-related target genes in animal models, whereas the regulation of HIF-1ɑ exerts its effects primarily through metabolic intervention [36].
HIF can affect gene expression through transcriptional regulation and cellular function through epigenetic mechanisms, which are closely associated with cellular dysfunction induced by the hypoxic microenvironment. First, DNA methylation can aberrantly activate HIF-1ɑ. In PASMCs od patients with PH, the promoter and enhancer regions of the superoxide dismutase 2 (SOD2) gene are aberrantly methylated at CpG islands (mediated by the DNA methyltransferases DNMT1 and DNMT3B), resulting in a significant downregulation of SOD2 expression [37]. Loss of function of SOD2 as a mitochondrial antioxidant enzyme triggers impaired mitochondrial superoxide (O2^–^) scavenging, resulting in oxidative stress and reduced hydrogen peroxide (H2O2) production (Fig. 2). Physiological reduction of H2O2 reduces its inhibitory effect on HIF-1ɑ, allowing for aberrant stabilization and activation of HIF-1ɑ under normoxic conditions [38]. Activated HIF-1ɑ promotes the overproliferation of PASMCs and vascular remodeling by upregulating the expression of pro-proliferative (VEGF) and anti-apoptotic genes, ultimately resulting in the progression of PH. Preclinical studies have also shown that cerium oxide nanoparticles (CeNPs) with SOD2/catalase mimetic activity can significantly improve hypoxia-induced PH, confirming the therapeutic potential of targeting oxidative stress [39]. Additionally, DNA methylation-induced SOD2 silencing and aberrant microRNAs (miR-124/335) regulate the pyruvate dehydrogenase kinase/pyruvate kinase type M2 (PDK/PKM2) pathway, resulting in a persistent pseudohypoxia. This maintains a pro-proliferative glycolytic phenotype when oxygen partial pressure is normalized and further contributes to PH progression. This is discussed under “Abnormalities of glucose metabolism” below.
Fig. 2SOD2 functional deficiency leads to oxidative stress [37]. Copyright 2025 Elsevier
Second, the SET domain is the hallmark catalytic domain of histone lysine methyltransferases (HKMTs), in the model of hypoxic PH mice, knockout of SETD2, a member of the SET domain family (SETD), in PASMCs significantly improved the PH phenotype, as evidenced by a 24% reduction in right ventricular systolic pressure (RVSP), a 28% reduction in mean pulmonary artery pressure (mPAP), and a 29% reduction in pulmonary artery medial thickness (PAMT) [40]. SETD enhance HIF signaling through a dual action. First, they directly increase the stability and DNA-binding capacity of the HIF-1ɑ protein by methylating it [41]. Second, they promote the transcription of HIF target genes, including VEGF, by catalyzing histone H3K36 trimethylation (H3K36me3) modification [42]. Furthermore, HIF-1ɑ transactivates the expression of DNA methyltransferases (DNMT3B), establishing a positive feedback loop of epigenetic-low oxygen signaling that further increases vascular remodeling [43]. These interactions ultimately result in the irreversible pulmonary arterial remodeling characteristic of PH, suggesting that targeting DNA/histone methylation can be a new strategy for intervention.
In summary, hypoxia-inducible factors (HIFs) not only directly regulate cellular processes such as cell proliferation, apoptosis, and metabolism, but also amplify hypoxic signals through interactions with pathways including metabolism, oxidative stress, and epigenetics, thereby forming a multi-level regulatory network that promotes the development of PH.
The Warburg effect, a phenomenon that resembles that of tumor cells, is demonstrated by PASMCs and PAECs, which exhibit a change in the pattern of glucose metabolism. This shift includes an enhancement of glycolytic pathways and a weakening of aerobic oxidation of sugars, even under aerobic conditions. In PH, abnormalities in gluconeogenesis begin with dysfunction of the mitochondrial oxygen-sensing system. First, hypoxia results in aberrant ROS signaling through epigenetic silencing of mitochondrial complex I and SOD2, triggering HIF-1ɑ stabilization, which drives metabolic reprogramming, including the switch from oxidative phosphorylation (OXPHOS) to glycolysis (Warburg effect) switch (Fig. 3) [17]. This process inhibits mitochondrial pyruvate metabolism by upregulating PDK and PKM2. In contrast, aberrant expression of the mitochondrial splitting protein Drp1 results in mitochondrial fragmentation, further impairing OXPHOS efficiency. Accompanying cytoplasmic calcium overload triggered by dysregulation of mitochondrial calcium homeostasis (MCUC dysfunction) synergistically activates pro-fission signaling, resulting in a glycolysis-dependent pseudo-hypoxic state. Epigenetic mechanisms (DNA methylation and miRNA aberrations) drive PH pathological progression through sustained inhibition of SOD2 and modulation of metabolic enzyme expression, ultimately resulting in aberrant proliferation, apoptosis resistance, and vascular remodeling of pulmonary artery cells [44].
Second, PDK and hexokinase (HK) are the key enzymes that regulate pyruvate metabolism. In a hypoxic microenvironment, upregulated PDK expression phosphorylates and inactivates pyruvate dehydrogenase (PDH), thereby inhibiting the entry of pyruvate into the tricarboxylic acid cycle (TCA cycle) for anaerobic glycolysis [45]. Increased glycolysis provides a rapid source of energy for the cells but also accumulates large amounts of lactic acid, resulting in intracellular acidosis and promoting microenvironmental acidification and vascular wall disruption. Additionally, hyperactivation of PDK promotes the Akt pathway, which is associated with the proliferation and migration of PASMCs and can increase PVR [46]. Studies have shown that as a rate-limiting enzyme of glycolysis, the overexpression of HK2 can promote inflammatory responses and metabolic reprogramming through the HIF-1ɑ/HK2/glycolysis axis, thereby exacerbating vascular remodeling in PH [47]. In addition, under hypoxic conditions, mitochondrial reactive oxygen species stabilize HIF-1ɑ protein by inhibiting HIF-1ɑ hydroxylation and activate glycolysis, which further drives the proliferation of PASMCs and vascular remodeling [19]. The mitochondrial localization of HK (HK1) also affects glycolysis-oxidative phosphorylation coupling, while abnormal HIF-2ɑ signaling in PAH may also aggravate the disease through metabolic disorders (such as lipid accumulation) [35, 48].
Additionally, lactate dehydrogenase A(LDHA)catalyzes the conversion of pyruvate to lactate and serves as a key enzyme in the final step of glycolysis. It is directly regulated by HIF-1ɑ. In PH patients and hypoxia models, increased LDHA expression leads to lactate accumulation, which promotes the proliferation of PASMCs and vascular remodeling [49]. Knockdown of LDHA can improve hypoxia-induced right ventricular dysfunction and vascular remodeling in mice, indicating its potential as a therapeutic target. Furthermore, LDHA enhances the migratory capacity of PASMCs by activating the Akt signaling pathway, thereby further exacerbating the progression of PH [50].
Fig. 3Mediators of Warburg metabolism in PH[17].(PDK: pyruvate dehydrogenase kinase, HK: hexokinase, PDH: pyruvate dehydrogenase). Copyright 2021 Elsevier
In hypoxic microenvironment-induced PH, abnormal lipid metabolism is mainly characterized by the dysregulation of fatty acid oxidation (FAO). This dysregulation not only affects pulmonary vascular cell function and vascular remodeling, but also is closely related to the maintenance of right ventricular function. Under hypoxic conditions, the transcriptional levels of FAO-related genes in the pulmonary arteries of PH patients are comprehensively upregulated [51]. In PH rats induced by SU5416-hypoxia (Su/Hx), the accumulation of long-chain fatty acids and triglycerides in the lungs and right ventricle was observed, accompanied by the upregulated expression of FAO rate-limiting enzymes CPT1a and CD36 [52]. In the hypoxia-SuHx model, inhibition of FAO can reduce ATP levels in the pulmonary arteries, increase mitochondrial mass, and decrease adventitial macrophage infiltration, thereby reversing vascular remodeling and hypoxic vasoconstriction [51]. In addition, under hypoxia, PAECs in PH upregulate FAO to generate β-hydroxybutyrate, which sensitizes the transient receptor potential vanilloid 4 (TRPV4) channel and accelerates their own proliferation and vascular remodeling in a Ca²⁺-dependent manner [53].
Right ventricular (RV) function is a key determinant of mortality in PH patients, and regulating FAO has become an important intervention direction for improving PH conditions and RV function. Among relevant interventions, trimetazidine (TMZ) inhibits FAO and switches metabolism to efficient glucose oxidation, reversing energy stress in the pulmonary vasculature and right ventricle, thus blocking the progression of pulmonary arterial hypertension [52]. Meanwhile, the carbonic anhydrase inhibitor acetazolamide can downregulate FAO and upregulate glucose oxidation to improve RV function [54].
In hypoxia-induced PH, abnormal amino acid metabolism is closely associated with disease progression. In rats exposed to a hypoxic environment, the levels of tryptophan, xanthurenic acid, and 3-indoleacetic acid were increased, while the levels of kynurenine, nicotinamide, and kynurenic acid were decreased [55]. Clinical studies further showed that the tryptophan metabolism pathway (KP) remains activated in patients with PH even during treatment with existing therapies. Elevated levels of circulating KP metabolites can predict the severity of PH, the response to PH treatment, and the survival of PH patients [56].
In addition to tryptophan, in the Su/Hx rat model, the levels of alanine, ornithine, aspartic acid, and downstream tricarboxylic acid (TCA) cycle intermediates were found to be decreased, while the levels of branched-chain amino acids (BCAAs) were increased [57]. Abnormal glycine metabolism also occurs under hypoxic conditions. HIF-1ɑ inhibits bolA-like protein 3 (BOLA3), leading to the loss of the glycine cleavage system and subsequent glycine accumulation. This accumulation further consumes one-carbon units, inhibits ATP synthesis, and activates mechanistic/mammalian target of rapamycin complex 1 (mTORC1) pro-proliferation signal, thereby inducing pulmonary vascular endothelial dysfunction and exacerbating PH [58]. Furthermore, hypoxia and mechanical stress jointly activate the YAP and TAZ signals in pulmonary arterial adventitial fibroblasts (PAAFs), promoting the catabolism of glutamine and serine and enhancing the biosynthesis of collagen, which contributes to the occurrence and development of PH [59].
Mitochondria are the primary site of energy production in the cell and an important source of ROS. Under a hypoxic microenvironment, the abnormal function of mitochondrial electron transport chain complexes I and III and the synergistic effect of NOX4 significantly increased ROS production [60]. The accumulation of mitochondrial ROS (mtROS) activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway, which promotes the migration of PAECs and the proliferation of PASMCs [61]. Under hypoxic conditions, the excessive increase of mtROS in PASMCs, accompanied by DNA damage, enables autophagy and promotes PVR [62].
NADPH oxidases (NOX) are a class of membrane-bound oxidases that catalyze the oxidation of NADPH to produce ROS. In PH, the expression activity of the NOX family members NOX2 and NOX4 is upregulated. In a model of chronic intermittent hypoxia, deletion of the NADPH oxidase subunit gp91phox (NOX2) significantly attenuates vascular remodeling and right ventricular hypertrophy [63]. Upregulation of NOX4 expression in PASMCs during chronic hypoxia increases NADPH oxidase activity, resulting in excessive ROS production, including superoxide (O2^–^) and hydrogen peroxide (H2O2) (Fig. 4) [64]. Moreover, excessive ROS induces cell membrane depolarization and increased calcium inward flow and enhances vasoconstrictor responses by inhibiting the current density of voltage-gated potassium channels (Kv) [65]. NOX4-mediated ROS promotes PASMCs proliferation and migration by oxidatively modifying signaling proteins, including PDGFRβ and activating the downstream Akt kinase pathway [66]. NADPH oxidase-derived ROS activate HIF-1ɑ, forming a positive feedback loop that further upregulates the expression of NOX4 and other pro-inflammatory factors, thereby exacerbating pulmonary artery endothelial dysfunction and inflammatory responses.
Fig. 4O2^–^ is converted to ROS, and NOX4 can result in overproduction of H2O2 [67]. (eNOS: expression of NO synthase, NOX: NADPH oxidases, SOD: superoxide dismutase). Copyright 2020 Multidisciplinary Digital Publishing Institute
Under normal physiological conditions, a well-established antioxidant defense system in the cell maintains redox balance. This system includes antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as antioxidants, including glutathione (GSH), which work together to scavenge excess ROS and maintain redox balance. In the hypoxic microenvironment, cells promote FoxO1 nuclear translocation by activating Adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) (2.5-fold increase in phosphorylation level) (40% increase in intranuclear FoxO1 content), which drives CAT expression [10, 68]. However, exogenous H2O2 (100 µM treatment for 6 h) activates the Akt pathway (60% increase in p-Akt), which results in FoxO1 nuclear export (35% decrease in intranuclear FoxO1), exhibiting a 45% and 30% reduction in CAT mRNA and protein, respectively, establishing a positive feedback loop of decreased antioxidant capacity [69]. This mechanism results in the breakdown of antioxidant defenses in the hypoxic microenvironment, making cells more susceptible to oxidative stress damage and promoting the onset and progression of PH.
Therefore, metabolic reprogramming not only provides energy support for cell proliferation, but also uses metabolites such as lactic acid, sphingosine, and glutamine as signaling molecules to activate pathways related to inflammation, proliferation, and apoptosis, thereby forming a metabolism-signaling coupling network.
Immune cell activation and release of inflammatory factors under hypoxic conditions result in PVR and endothelial cell dysfunction. Macrophages are classified into M1 and M2 types, with M1 macrophages producing pro-inflammatory factors, including TNF-ɑ, IL-6, IL-1β, and ROS, to exacerbate endothelial injury early in the onset of PH. Moreover, M1 macrophages mediate defense mechanisms against invading pathogens, including bacteria and viruses [70]. M1 macrophages release IL-1β through NLRP3 inflammasomes, which promotes the proliferation and migration of PASMCs proliferation and migration. Additionally, inhibition of Bruton tyrosine kinase (BTK) could alleviate PH by regulating macrophage recruitment and polarization, which can be a potential therapeutic strategy for treating PH[71].
During the PH progression phase (14−28 days), M2-type macrophages gradually dominate, promoting abnormal proliferation of PASMCs and vaso-occlusive remodeling through paracrine effects [72]. The secretion of the pro-fibrotic factors, including transforming growth factor β (TGF-β), PDGF, CCL18, and the angiogenic factor VEGF, drives vascular smooth muscle proliferation and extracellular matrix deposition. This process is involved in promoting anti-inflammatory responses. M2-type and PASMCs form a positive feedback loop through CCL2-CCR2/CCL5-CCR5 signaling and amplify the pro-proliferative effects [33]. Furthermore, the imbalance between M1 and M2 polarization contributes to the PH development. In an experimental model of PH, macrophages in male mice were polarized towards a pro-inflammatory M1 or pro-fibrotic M2 phenotype, resulting in an imbalance in the M1 macrophage/M2 macrophage ratio that directly contributed to the PH phenotype [73].
T cells play an important role in immune regulation. In patients with hypoxia-induced PH, T cell depletion is a phenomenon characterized by the gradual loss of T cell proliferation and cytokine secretion in response to long-term antigenic stimulation, including chronic infection, tumor, or autoimmune disease. This is evidenced by the elevated expression of immune checkpoint molecules, including surface inhibitory receptors (programmed death receptor 1 (PD-1), cytotoxic T cell-associated protein 4 (CTLA-4), and other immune checkpoint molecules [74]. Hypoxia can activate PD-1 expression in T cells by upregulating HIF-1ɑ. HIF-1ɑ not only directly induces PD-1 expression but also inhibits the degranulation capacity of CD8⁺ T cells, resulting in a multiplicity of immunosuppressive effects [75]. Activated CD8⁺ T cells secrete the inflammatory factor TNF-α, which inhibits PDH activity in PASMCs. This inhibition leads to mitochondrial hyperpolarization, reduced mROS production, downregulated Kv1.5 channel expression, increased intracellular calcium ion concentrations, and activated nuclear factor of activated T-cells (NFAT). These changes induce aberrant proliferation and apoptosis resistance in PASMCs, resulting in the PH phenotype [29, 76]. The expression of PD-1 and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) on the surface of CD4 + and CD8 + T cells was found to be significantly elevated in the peripheral blood mononuclear cells of patients with PH (including idiopathic PH, congenital heart disease-associated PH, and other subtypes), which was positively correlated with the severity of the disease. It has been hypothesized that PD-1 overexpression can result in T-cell depletion, which in turn is involved in the immunopathological process of PH[77].
The hypoxic microenvironment reshapes the function of immune cells, forming a pro-inflammatory and pro-remodeling immune microenvironment, the immune-inflammatory response, together with HIFs and oxidative stress, forms an “inflammation-HIF-ROS” positive feedback mechanism. Moreover, immune cells (including M1/M2 macrophages, NETs, and T cells) and PASMCs as well as PAECs form a complex cell-cell communication network, which collectively drives inflammation and remodeling.
Apoptosis is an important mechanism for maintaining tissue homeostasis under normal physiological conditions; however, during the pathological process of PH, the hypoxic microenvironment can regulate apoptosis-related pathways in a multidimensional manner. This can result in apoptosis resistance or aberrant survival of PASMCs and PAECs, which in turn drives PVR and the progression of PH. First, hypoxia can affect apoptosis in PASMCs by activating mitochondria-dependent apoptotic pathways. In patients with PH and hypoxia-induced PAECs, phosphorylation of the signal transcriptional activator signal transducer and activator of transcription 3 (STAT3) tyrosine 705 site (STAT3Tyr705), which promotes mitochondrial division and inhibits apoptosis in PAECs by upregulating the dynamin-related protein 1(DRP1), results in PVR [78, 79]. Second, transient receptor potential melastatin 7 (TRPM7) channels, which are downregulated in hypoxic PH, could over-activate the MAPK signaling pathway MEK/ERK, which promotes the proliferation of PASMCs and inhibits apoptosis [80]. Additionally, bone morphogenetic protein receptor type 2 (BMPR2), a transmembrane serine/threonine kinase receptor and a member of the TGF-β superfamily, plays a key role in regulating cell proliferation, apoptosis, and vascular homeostasis [81]. BMPR2 activates the downstream SMAD1/5/8 pathway by binding bone morphogenetic proteins, promotes the expression of the anti-proliferative genes ID1 and ID2, and inhibits PASMC’s proliferation. Moreover, ID2 expression inhibits the proliferation of PASMCs and ensures the survival of PAECs [82]. In patients with PH, mutation or reduced expression of the BMPR2 gene results in impairment of the signaling pathway, triggering aberrant apoptosis of PAECs and over-proliferation of PASMCs and promoting vascular remodeling (Fig. 5) [83].
Fig. 5A link between BMPR2-signaling and mitochondrial function [83]. (Loss of BMPR2 in PAECs during normoxia enhances mitochondrial biogenesis, driven by p53 PGC1ɑ, NRF2 and TFAM, increases mitochondrial membrane potential and ATP, and glycolysis, leading to fission and causing a pro-inflammatory state via Interleukins such as IL8. The increase in TFAM appears to partially mitigate the increase in IL8. Loss of BMPR2 during reoxygenation after hypoxia impairs p53-dependent regulation of PGC1ɑ and TFAM, reduces mitochondrial membrane potential and ATP and causes mitochondrial fission and mitochondrial DNA deletion, inducing PAECs apoptosis leading to loss of microvessels and aberrant proliferation of SM-like cells. Both inflammation and EC apoptosis contribute to pulmonary arterial hypertension (Red arrows indicate up or down regulation; black arrows, sequalea). Copyright 2015 Cell Press
Autophagy is a complex process in which cells transport aged or dysfunctional organelles to lysosomes for degradation. Through this process, cells obtain new building blocks and energy, which are essential for cellular renewal and maintaining cellular homeostasis [84]. In PH, hypoxia induces altered levels of autophagy, and the effects exhibit significant cell-type-specific and dual regulatory properties. Moderate autophagy can be cytoprotective, maintaining the stability of the intracellular environment by removing damaged organelles and protein aggregates. However, either excessive autophagy or defective autophagy can result in cellular dysfunction. In PAECs, moderate autophagy inhibits endothelial-mesenchymal transition by degrading EndMT key transcription factors (Snail and Twist), thereby alleviating vascular remodeling [85]. However, in PASMCs, over-activation or inhibition of autophagy (ROS-autophagy imbalance due to TIGAR deficiency) promotes cell proliferation and migration and exacerbates PVR. Hypoxia upregulation of circ-calm4 drives autophagy-dependent proliferation by enhancing Beclin1 transcription through binding with Purb [86].
Furthermore, the AMPK pathway plays a key role in regulating hypoxia-induced mtROS inhibits autophagy and enhances apoptosis resistance in PASMCs, whereas it exerts a protective effect by activating autophagy in aortic smooth muscle cells. AMPK ɑ1 maintains the survival of PASMCs by inducing autophagy, and AMPK ɑ2 inhibits apoptosis by regulating MCL-1, highlighting the cross-regulation of autophagy and apoptosis [87]. Additionally, the transcriptional regulator phytochrome interacting factor 1 (PIF1) inhibited apoptosis in PAECs by promoting autophagy under hypoxia. Studies have suggested that chronic hypoxia upregulates PIF1 expression and enhances autophagic activity (LC3-II increase and P62 degradation), thereby protecting PAECs from apoptosis, while inhibiting autophagy exacerbates apoptosis [85].
In the hypoxic microenvironment, HIF-1ɑ and oxidative stress collectively maintain the abnormal proliferation of PASMCs and PAECs by regulating apoptosis and autophagy, thereby promoting pulmonary vascular remodeling.
Mechanical stress is the distribution of internal resistance within an object due to externally applied forces (tension, compression, or shear), which primarily includes the following three (1) Fluid shear tangential friction generated by blood flow on the surfaces of PAECs. (2) Tensile Stretch: the tensile force on PASMCs and PAAFs when the vessel wall expands due to elevated pressure. (3) Extracellular Matrix (ECM) Stiffness: localized mechanical resistance due to hardening of the ECM, which affects cell adhesion and migration (Fig. 6) [88]. Consequently, an increase in blood flow velocity increases shear stress, whereas vasoconstriction and dilatation cause changes in tensile or compressive stress.
Fig. 6Intracellular and intercellular signaling pathways in pulmonary arterial cells regulated by fluid shear stress, stretch, hypoxia and extracellular matrix (ECM) stiffening [88]. Copyright 2021 Springer Link
In the hypoxic environment, activation of the YAP/TAZ signaling pathway and mechanical stress formation act synergistically to drive the pathological process of PH. First, hypoxia inhibits Hippo kinase activity, reduces YAP/TAZ phosphorylation, and promotes nuclear translocation by upregulating HSP110 expression or activating pathways, including TGF-β1/ROCK[22]. Meanwhile, hypoxia-induced PVR (smooth muscle cell proliferation and collagen deposition) increases vascular wall mechanical stress, the latter of which further activates YAP/TAZ through integrin-β3/FAK signaling and RhoA-ROCK-dependent cytoskeletal reorganization [89]. The activated YAP/TAZ binds to TEA domain transcription factor 4 (TEAD4) to drive pro-fibrotic genes (collagen and fibronectin), accelerating ECM hardening, and providing precursors for collagen synthesis through metabolic reprogramming (enhanced glutamine/serine catabolism). This forms a positive feedback loop of “hypoxia-mechanical stress-YAP/TAZ-ECM hardening”[59]. Furthermore, YAP/TAZ directly increases pulmonary vascular resistance by promoting the proliferation and migration of PASMCs and inhibiting autophagy. It affects cardiac compensation through right ventricular cardiomyocyte hypertrophy and ECM remodeling, ultimately progressing to PH[90].
In hypoxic environments, mechanical stress in pulmonary arteries drives the PH development through multidimensional activation of the RhoA/ROCK pathway. In hypoxia, enhanced mechanical stress first directly stimulates membrane depolarization in PASMCs, activates RhoA, and promotes its binding to ROCK. This stimulation activates myosin light chain phosphorylation through inhibition of myosin light chain phosphatase, regulates assembly and depolymerization of actin filaments, elevates cytosolic calcium sensitivity, and affects cytoskeletal stability and cellular morphology, results in abnormal vasoconstriction [91]. Meanwhile, mechanical stress increases the calcium inward flow function of TRPC1/TRPC6 channels by upregulating HIF-1ɑ expression, activating RhoA/ROCK-dependent cell proliferation signaling, and promoting migration of PASMCs and thickening of pulmonary artery walls (Fig. 7) [92]. In addition, mechanical stress promotes sustained positive feedback through oxidative stress feedback loops (superoxide-dependent RhoA activation) and epigenetic regulation (competitive inhibition of miR-141-targeted silencing of RhoA by lncRNA-SMILR) to amplify the vascular remodeling effect [92–94]. This mechanical-chemical signaling interaction ultimately results in elevated pulmonary vascular resistance and right ventricular hypertrophy. This highlights the role of the RhoA/ROCK pathway as a central hub of mechanical stress transduction in hypoxic PH and provides a theoretical basis for targeted inhibition of this pathway (ROCK inhibitors and statins).
Fig. 7Proposed mechanism for the role of PASMCs-Piezo1 (a mechanically sensitive ion channel) in the development and progression of PH[92]. Copyright 2025Lippincott Williams and Wilkins Ltd
Therefore, mechanical stress, together with HIFs and the YAP/TAZ pathway, not only alters cell morphology, but also regulates processes such as proliferation, contraction, and apoptosis through signaling pathways, forming a structure-function coupling network that promotes the development of PH.
MicroRNAs, as a class of small non-coding RNA molecules, negatively regulate gene expression by binding to the 3’ untranslated region of target gene mRNAs and participate in cell proliferation, apoptosis, and inflammation [84, 95, 96]. A recent study has confirmed that the combination of miRNA signatures with N-terminal pro-B-type natriuretic peptide (NT-proBNP), age and gender is superior to the use of NT-proBNP or miRNA alone in the subclassification of PH subtypes [97]. In the peripheral circulating exosomes of patients with HIV-associated pulmonary hypertension (HIV-PH), the expressions of miR-32-5p and miR-92b-3p are upregulated, while the expressions of miR-5571 and miR-4670 are downregulated. These changes are key molecular events driving pulmonary vascular remodeling and PH. In hypoxia-induced PH, miRNAs drive pathological progression through multi-targeted hypoxia inhibits miR-328 expression, deregulates its inhibitory effects on L-type calcium channel ɑ1C (LTCC α1C) and insulin-like growth factor 1 receptor (IGF1R), and exacerbates calcium-dependent vasoconstriction and PASMCs proliferation [98]. MicroRNA-210 impairs mitochondrial bioenergetics and reduces mtROS production by targeting and inhibiting subunits of the mitochondrial electron transport chain (NDUFA4, SDHD, COX10). This weakens the negative feedback of HIF-1ɑ, continuously amplifies hypoxia-induced proliferation of PASMCs and pulmonary vascular remodeling, and ultimately promotes the development of PH[99]. Prophylactic or therapeutic administration of miR-29a-3p can significantly reduce pulmonary arterial pressure and right ventricular hypertrophy index in rats with hypoxic pulmonary hypertension, and improve pulmonary vascular remodeling [100]. In addition, hypoxia inhibits miR-146b, thereby inducing the expression of pro-inflammatory IL-6 and CCL2 (MCP-1). This promotes ventricular inflammatory infiltration, imbalance between cell apoptosis and hypertrophy, as well as ventricular dysfunction [101]. This contributes to the further deterioration of right heart function [100, 101]. In summary, hypoxia interferes with calcium homeostasis, proliferation-apoptosis balance, and inflammatory response through a dynamic regulatory network of miRNAs, ultimately triggering pulmonary artery remodeling and right heart failure, constituting the core pathological mechanism of PH.
Long non-coding RNAs (lncRNAs) are defined as transcripts with a length exceeding 200 nucleotides and no protein-coding potential [102]. In the PH model of Sprague-Dawley rats, lncRNA has been confirmed to be closely associated with hypoxia and may be involved in disease progression by regulating vascular remodeling [103]. In PH, the expression of lncRNA-maternally expressed gene 3 (MEG3) was significantly downregulated (based on assay data from lung tissues and pulmonary arteries of patients with PH). Its mechanism of action primarily acting as a competing endogenous RNA to adsorb long-stranded RNAs (miRNAs), including miR-34a-3p, and deregulating the target gene DUSP1 inhibition, thus promoting the proliferation and migration of PASMCs [104]. In hypoxic PH, the level of lncRNA-MEG3 is significantly increased in the cytoplasm of PASMCs. The upregulation of lncRNA-MEG3 binds to miR-328-3p in the cytoplasm, which ultimately leads to the expression of IGF1R and promotes the development of PH[98]. Platelet-derived growth factor-BB (PDGF-BB)-upregulated lncRNA VELRP promotes excessive proliferation of PASMCs and pulmonary vascular remodeling through the WDR5/CDK signaling pathway. In a rat PH model, specific knockdown of VELRP can significantly reduce right ventricular systolic pressure and alleviate vascular remodeling, providing a new lncRNA therapeutic target for PH[105]. The long non-coding RNA TYKRIL is significantly upregulated in PASMCs from patients with PH and hypoxic rat models. Mechanistically, TYKRIL directly binds to p53 and enhances its transcriptional activity. This strengthens the inhibitory effect of p53 on the PDGFRβ promoter, thereby downregulating the PDGFRβ-PI3K/AKT pathway and reducing the expression of pro-proliferative proteins such as PCNA and Cyclin A/E. Ultimately, it significantly reduces PASMC proliferation and pulmonary vascular remodeling, decreases right ventricular systolic pressure, and effectively delays the progression of PH[106].
CircRNAs are covalently closed circular non-coding RNA molecules formed by reverse splicing, which are highly stable and tissue-specific. circRNAs can be involved in disease regulation through adsorption of miRNAs, binding of functional proteins, or modulation of gene translation [107]. In hypoxic PH, circRNAs drive PVR and dysfunction through multidimensional (1) signaling pathway regulation, including circ-Ntrk2, which activates the TGF-β1/p38 MAPK pathway to promote vascular remodeling through sponge adsorption of miR-296-5p to deregulate its inhibition of TGF-β1[107, 108]. Additionally, circ-myh8 recruits histone acetyltransferase KAT7 to the HIF1ɑ promoter region, increases the H3K5 acetylation levels, and significantly elevates the HIF1ɑ expression to accelerate the abnormal proliferation of PASMCs (Fig. 8) [108]. Recent studies have found that 5-methylcytosine (m⁵C)-modified circular RNA CCNL2 (circ-CCNL2) is significantly upregulated in hypoxic PH. It directly binds to the RNA-binding protein, fragile X mental retardation syndrome-related protein 2 (FXR2), and then inhibits FXR2-mediated transcriptional activation of pro-proliferative genes. This blocks the excessive proliferation of PASMCs and collagen deposition, ultimately alleviating pulmonary vascular remodeling and reducing right ventricular pressure. This suggests that the synergistic effect between RNA modifications (m⁵C) and protein binding may serve as a novel intervention target [109]. (2) Cell proliferation and metabolic circPMS1 promotes the abnormal proliferation of PASMCs, endothelial cells, and pericytes under hypoxia through different miRNA axes, including miR-432-5p/DEPDC1 and miR-433-3p/MXI1. Furthermore, circNAP1L4 regulates the hyper-enforcer of the core gene of glycolysis, HK2, by binding to the host protein’s activity and inhibits the glycolysis-proliferation coupling process in PASMCs [110]. (3) Cell death and homeostatic intra-nuclear circ-calm4 induces iron death through circR-loop inhibition of COMP gene expression [111]. circSSR1 promotes SSR1 protein translation through m6A modification to activate endoplasmic reticulum stress-dependent pyroptosis [111, 112]. Moreover, circ-calm4 enhances Beclin1 super-enhancer activity by interacting with Purb proteins, which in turn promotes autophagy-associated vascular remodeling [86]. (4) Ion channels and post-translational circNFXL1 restores potassium channel function and alleviates calcium overload through the miR-29b-2-5p/Kv2.1 axis [113]. CircGUCY1A2 inhibits phenotypic switching in PASMCs by blocking the O-glycosylation modification of COL3A1 [114]. The above mechanisms suggest that circRNAs intervene in the hypoxic PH process at multiple levels through epigenetic regulation, signaling network integration, and post-translational modifications, providing potential targets for developing novel therapeutic strategies.
The aforementioned mechanisms indicate that circRNAs interfere with the progression of hypoxic pulmonary hypertension through multiple levels, such as epigenetic regulation, signal network integration, and post-translational modification, providing potential targets for the development of novel therapeutic strategies. However, current research still has significant firstly, most mechanistic studies focus on the linear regulatory relationships of a single circRNA, and the regulatory networks formed between circRNAs have not yet been clarified. In particular, there is a lack of systematic functional analysis of special subtypes such as chromatin-associated circRNAs (ca-circRNAs). Secondly, the regulation of circRNA functions by RNA modifications (e.g., m⁵C, m⁶A) is cell-type-specific, and the differences in their roles among different cell types of pulmonary blood vessels have not been clarified. Finally, some newly discovered mechanisms (such as the regulation of m⁵C modification of circ-CCNL2) lack verification in clinical samples, making it difficult to determine their correlation with disease progression.
Fig. 8Circ-myh8 alters histone modification patterns by recruiting KAT7 to the promoter region of the HIF-1ɑ gene as a modular scaffold, thereby increasing H4K5 acetylation in its promoter region, ultimately altering HIF-1ɑ expression and promoting proliferation and PVR in PASMCs [108]. Copyright 2023 Wiley-Blackwell Publishing Ltd
It can be seen from this that ncRNAs, as post-transcriptional regulatory nodes, integrate mechanisms such as HIFs, metabolism, oxidative stress, immune signals, and epigenetics to form a multi-level regulatory network, thereby promoting the development of PH.
Gut flora metabolites are active molecules (short-chain fatty acids, SCFAs; trimethylamine N-oxide, TMAO; asymmetric dimethylarginine, ADMA) produced by gut microorganisms through the metabolism of dietary or host endogenous substances. The effects of microbe-host interactions on PH have recently become an emerging area of research. Existing research has demonstrated that in a hypoxic microenvironment, intestinal flora dysbiosis triggers immune dysregulation and systemic inflammation by altering circulating microbial metabolites (such as short-chain fatty acids, trimethylamine N-oxide, bile acids, and tryptophan), thereby promoting the development of pulmonary vascular diseases [23]. These metabolites affect pulmonary immune regulation and vascular remodeling through the “gut-lung axis”: (1) Hypoxia induces the proliferation of ADMA-producing bacteria (Streptococcus), which exacerbates pulmonary vasoconstriction through inhibition of nitric oxide synthase (NOAS) and activation of NLRP3 inflammasome by TMAO. This promotes the release of inflammatory factors, including IL-1β, IL-18, NLRP3, IL-18, and other inflammatory factors, triggering an inflammatory response and promoting vascular remodeling [115]. (2) SCFAs such as butyrate and propionate have anti-inflammatory effects. A reduction in their levels impairs the function of regulatory T cells, promoting pulmonary inflammatory responses and vascular remodeling [23]. Second, hypoxia inhibits beneficial bacteria (Roseburia), decreasing short-chain fatty acids and impairing their anti-inflammatory and endothelial protective effects.
However, current preliminary observations reveal that the role of the intestinal multi-kingdom microbiota (including bacteria, archaea, and fungi) in PH remains unclear. For instance, although metagenomic analysis indicates an imbalance in the multi-kingdom microbiota, the specific mechanisms of their contribution (such as the role of fungi or viruses) have not yet been fully elucidated and require further verification [116, 117]. Recent studies suggest that intestinal microbiota-targeted intervention strategies (microbiota transplantation, selective antibiotics, and metabolic pathway-targeted modulators) hold diagnostic and therapeutic potential [118].
Therefore, gut microbiota metabolites promote pulmonary inflammation and vascular remodeling by activating inflammasomes to release inflammatory factors and interacting with immune cells. Meanwhile, these metabolites may further exacerbate the pathological process of PH by influencing the HIF signaling pathway and oxidative stress.
DNA methylation is an epigenetic modification process in which a methyl group is added to the cytosine at the 5’ end of the CpG dinucleotide in the DNA molecule, catalyzed primarily by DNA methyltransferases (DNMTs), and regulates cellular function by silencing or activating gene expression. Under hypoxic conditions, aberrant DNA methylation drives the PH development through multiple key gene-targeted methylation (1) Hypoxia induces alterations in cytosine methylation in the IGF-1 promoter region through histone deacetylases (HDACs), activating its downstream PI3K/AKT signaling and accelerating PVR [119]. (2) Lung vascular endothelin B receptor promoter demethylation results in upregulation of its expression and activation of NADPH oxidase (Nox1/4)-dependent oxidative stress pathway, enhancing vasoconstriction and proliferation of PASMCs [120]. (3) PTEN gene promoter hypermethylation inhibits its expression and reverses the inhibitory effect on PASMCs proliferation [121]. (4) piRNA-63,076 promotes aberrant proliferation of PASMCs by methylating the acyl-coenzyme A dehydrogenase promoter [122]. Prenatal/perinatal programming effects indicate that early hypoxic exposure creates a persistent “epigenetic memory” through altered DNA methylation profiles (abnormalities in inflammation- and proliferation-related genes) in PASMCs, further exacerbating PH susceptibility to hypoxia in adulthood [123]. Regarding intervention potential, DNMT (5-Aza-dC) or HDAC inhibitors can inhibit PVR and right heart hypertrophy by reversing methylation aberrations and restoring target gene function (PTEN and IGF-1)[124, 125]. Accordingly, hypoxia integrates inflammatory, oxidative stress, and cell proliferation signals through a dynamic regulatory network of local or global DNA methylation, which is a central epigenetic mechanism of the PH process and a potential therapeutic target.
Histone modification is an important mechanism of epigenetic regulation, altering chromatin structure through methylation and acetylation, thereby regulating gene expression. First, hypoxia upregulates histone methyltransferase SETDB1 through a HIF2ɑ-dependent pathway, resulting in increased levels of H3K9 trimethylation, which triggers apoptosis, senescence, and endothelial-mesenchymal transition of pulmonary arterial microvessel endothelial cells, and promotes PVR [126]. Second, cyclic RNA circ-myh8 recruits histone acetyltransferase KAT7 to the HIF1ɑ promoter region, increases the level of H4K5 acetylation, activates HIF1ɑ transcription, and promotes aberrant proliferation of PASMCs [127]. The histone methyltransferase SETD2 upregulates the m6A methylase METTL14 by catalyzing H3K36 trimethylation, forming an epigenetic cascade that exacerbates vascular remodeling [128]. Besides, hypoxia induces H3K4 trimethylation and DNA hypomethylation in the promoter region of the ET-1 gene in an intrauterine growth restriction model through a trans-representational epigenetic mechanism, which increases PH susceptibility by sustained high expression of ET-1 in progeny endothelial cells [123]. Furthermore, newly identified histone lactonylation modifications can further promote hypoxia-associated vascular inflammatory responses by interfering with m6A methylase activity and immune cell function [123]. These histone modification abnormalities ultimately result in elevated pulmonary vascular resistance and PH development by modulating the HIF pathway, cell proliferation/apoptosis balance, and vasoactive factor expression.
RNA modifications are chemical modifications that occur in post-transcriptional RNA molecules, including N6-methyladenosine (m6A) and 8-oxoguanine (8-oxoguanine), that affect gene expression by modulating RNA stability, translation efficiency, or protein interactions. Under hypoxic conditions, RNA modifications drive the pathological process of PH through multiple first, in terms of m6A methylation, hypoxia downregulates the methyltransferase METTL3, which reduces the level of m6A modification of PTEN mRNAs, resulting in a decrease in their stability, and promotes the charring and abnormal proliferation of PASMCs (Fig. 9) [129]. Furthermore, the m6A-reading protein YTHDF1 promotes the translation of MAGED1 mRNA by recognizing its m6A site, activating PASMCs proliferation and vascular remodeling [130]. Second, in terms of circular RNA regulation, hypoxia induced high expression of circ-myh8, which promotes histone H4K5 acetylation by recruiting histone acetyltransferase KAT7 to the HIF-1ɑ promoter region, enhances HIF-1ɑ transcription, and increases vascular remodeling [127]. In terms of oxidative modification of tRNA fragments, hypoxia induced 8-oxo-modification of tRF-1-AspGTC (5o8G tRF) promotes PASMCs proliferation and apoptosis resistance by inhibiting the expression of WNT5A and CASP3 and activating the BMPR2-ROS pathway (Fig. 10) [131]. In conclusion, hypoxia multidimensionally regulates abnormal vascular smooth muscle function and endothelial dysregulation through RNA modification, ultimately resulting in vascular pathological remodeling in PH, providing a potential therapeutic strategy for targeting key molecules, including METTL3, YTHDF1, or circRNA.
Fig. 9M6A methyltransferase METTL3 is involved in hypoxia-induced PASMCs pyroptosis, PTEN is a downstream target of METTL3, and IGF2BP2 directly binds to the m6A site on PTEN mRNA and enhances PTEN mRNA stability [129]. Copyright 2024 Wiley-Blackwell Publishing Ltd
Fig. 10Hypoxia induces 5o8G tRF modification, inhibits WNT5A and CASP3 expression, activates the BMPR2-ROS pathway, and promotes PASMCs proliferation and apoptosis resistance [131]. Copyright 2024 Lippincott Williams and Wilkins Ltd
In summary, hypoxia-induced HIF-1ɑ not only affects gene expression through transcriptional regulation, but also stabilizes its own expression via epigenetic mechanisms, forming a positive feedback loop that further promotes the development of PH. Additionally, metabolic reprogramming interacts with epigenetic regulation in the hypoxic by altering the expression and activity of metabolic enzymes, it affects the modification patterns of DNA and histones, thereby driving cell proliferation and vascular remodeling. Meanwhile, non-coding RNAs also participate in the regulation of epigenetic mechanisms, influencing the HIF signaling pathway and oxidative stress response, which further exacerbates the pathological process of PH. These multi-level interactions constitute a complex regulatory network in the hypoxic microenvironment, collectively driving the occurrence and progression of PH.
The Transient Receptor Potential (TRP) channel superfamily is a large group of non-selective cation channels, which includes multiple subfamilies such as TRPC, TRPV, and TRPM. It plays a core role in sensing a variety of extracellular and intracellular stimuli (hypoxia, mechanical stress, oxidative stress, inflammatory mediators) and mediating Ca²⁺ influx [132]. In hypoxic PH, the expression and function of multiple TRP channel subtypes undergo significant changes. By inducing sustained intracellular Ca²⁺ overload, TRP channels become a key link in promoting pulmonary vasoconstriction and remodeling.
The TRPC subfamily, especially TRPC1 and TRPC6, are core channels involved in hypoxia-induced Ca²⁺ overload in PASMCs. Studies have shown that long-term hypoxia significantly upregulates the expression and activity of TRPC1 and TRPC6 in PASMCs, thereby promoting PASMCs proliferation [133]. In contrast, triple knockout of TRPC1/3/6 significantly weakens hypoxia-induced pulmonary arterial contractile responses, and reduces right ventricular pressure and vascular remodeling [134]. Additionally, experiments have confirmed that sildenafil can directly transcriptionally inhibit TRPC1/TRPC6 by activating PPARγ, which reduces Ca²⁺ influx and PASMCs proliferation, thereby alleviating hypoxia-induced PH and right ventricular hypertrophy in newborn rats [135].
In PH, TRP channels not only mediate calcium influx but also act as key signal integrators, closely interacting with multiple core pathological mechanisms in the hypoxic microenvironment to collectively influence disease progression.
In terms of oxidative stress, NOX-regulated ROS production can affect the expression and activity of TRPC channels, leading to changes in Ca²⁺ concentration in PASMCs and inducing vasoconstriction [136]. Additionally, recent studies have shown that in chronic hypoxia-induced PH, the expression of NOX1 and NOX4 in right coronary artery smooth muscle cells is significantly upregulated, resulting in a sustained increase in intracellular H₂O₂ levels. This activates the TRPM2 channel, enhances Ca²⁺ influx, thereby significantly augmenting the coronary artery’s contractile response to serotonin and inducing a metabolic shift in coronary artery smooth muscle cells toward glycolysis. Inhibition of NOX1/4 or TRPM2 can ROS and Ca²⁺ signals, alleviate vasoconstriction and right ventricular pressure, and delay disease progression. These findings suggest that the NOX1/4-TRPM2-ROS-Ca²⁺ axis plays an important role in PH-related coronary artery remodeling and coronary artery disease [49].
As an upstream core regulator, HIF directly promotes the expression of TRP channels. Under hypoxic conditions, HIF-1ɑ is stabilized and activated, which induces the upregulation of BMP4 expression, promotes increased transcription of TRPC channels in PASMCs, leads to enhanced calcium influx, and thereby participates in the processes of pulmonary vasoconstriction and remodeling [137]. In addition, inhibiting the RhoA/ROCK pathway can reduce HIF-1ɑ levels, diminish its transcriptional activation of TRPC channels, thereby attenuating calcium influx in PASMCs and pulmonary vascular remodeling, and alleviating hypoxia-induced PH[138].
Regarding cell proliferation, TRPM7 has been identified as a novel negative regulator in the pathogenesis of PH. Downregulation of TRPM7 relieves its negative regulation on the MEK/ERK pathway, leading to excessive proliferation of pulmonary artery smooth muscle cells, inhibition of their apoptosis, promotion of pulmonary vascular remodeling, and subsequent exacerbation of PH[80]. Furthermore, the pro-inflammatory transcription factor NF-κB/p65 and the anti-proliferative transcription factor PPARγ inversely regulate TRPC6 expression through competitive binding to the TRPC6 promoter. This process determines cellular Ca²⁺ signaling, proliferation, and migration phenotypes, and is directly involved in the occurrence and development of PH[139]. In terms of cell apoptosis, TRPC4 promotes the expression of Susd2, thereby activating hypoxia-induced apoptosis of pulmonary artery endothelial cells. Inhibiting TRPC4 by blocking the Susd2 signaling pathway can reduce pulmonary artery endothelial cell apoptosis in animal models and improve hypoxic pulmonary hypertension, indicating that this pathway may serve as a potential therapeutic target for PH[140].
Despite the above validated achievements in TRP channel research, some aspects remain in the preliminary discovery stage. For instance, the upstream regulatory mechanisms of novel regulators such as TRPM7 and their interaction networks with other signaling molecules are yet to be clarified. Whether more complex synergistic or antagonistic effects exist between different TRP channel subtypes is currently only in the initial exploration phase, with no definitive conclusions. Moreover, the vast majority of current studies on TRP channels are still in the preclinical stage. Although multiple promising targets such as TRPM2 and TRPC4 have been identified, there is a lack of clinically available drugs with high specificity and low side effects. How to translate basic research findings into safe and effective therapies is one of the greatest challenges at present.
To address PH pathogenesis in the hypoxic microenvironment, multiple drugs have been applied in the clinic or are in the research stage. Future drug design can require co-target pathways, including HIF-ɑ, non-coding RNA regulation, and epigenetic regulation to mediate metabolic reprogramming, redox imbalance, immunoinflammation, modulation of cell death patterns, mechanical stress, and microbial-host interactions. This will enable the achievement of a multidimensional inhibition of PVR.
In recent years, targeted therapy against HIF-1ɑ has shown promising prospects. 2-methoxyestradiol (2-ME-2), an estrogen metabolite, is a HIF-1ɑ inhibitor that can reduce the proliferation of human PASMCs, slow down the progression of PH, and its efficacy exhibits gender differences [141]. Prostaglandin E1 (PGE1) can reduce cell apoptosis by regulating HIF-1ɑ and improve the homing of mesenchymal stem cells in PH[142]. Another emerging strategy involves drugs targeting epigenetic modifications. The use of the HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) can restore the number and function of Foxp3⁺ regulatory T cells (Treg) in lung tissue and peripheral blood, thereby significantly alleviating pulmonary vascular remodeling, reducing right ventricular pressure, and ultimately reversing experimental PH[143].
Endothelin receptor bosentan and andrisentan are the commonly used medications in the treatment of PH at present. They block the binding of ET-1 and its receptor, reduce vasoconstriction and early vascular occlusive lesions, and improve PVR. Their ability to intervene early in the hypoxic phase can reduce the pulmonary vascular occlusion index and slow down the progression of PH[144].
Prostacyclin iloprost directly dilate pulmonary artery vasculature by agonizing IP receptors, significantly lowering mPAP and right ventricular systolic pressure, reducing right heart afterload and improving right ventricular systolic function [145]. Novel nanoparticle delivery systems (intratracheal administration of beraprost) can enhance the local action of the drug, prolong the anti-proliferative and pro-apoptotic effects, and reduce systemic side effects [146]. Prostacyclin analogs have been demonstrated to significantly reduce the risk of death or complications in patients with PH in large clinical trials. They have been recommended as a core therapeutic option, particularly in connective tissue disease-associated PH.
In hypoxaemic patients with PH, mechanical ventilation and oxygen therapy, as the core measures to ameliorate hypoxia, need to be tailored to balance therapeutic efficacy and potential risks. Mechanical ventilation reduces hypoxic pulmonary vasoconstriction (HPV) by opening the alveoli with positive pressure and optimizing the ventilation/blood flow ratio, thus reducing pulmonary vascular resistance. However, its application requires following the principle of lung protection (small tidal volume and limited plateau pressure) to avoid excessive airway pressure that inhibits venous return or induces ventilator-associated lung injury. Oxygen therapy directly inhibits HPV and alleviates HIF-1ɑ-mediated vascular remodeling by increasing the partial pressure of blood oxygen. However, the target oxygen saturation (90%−94%) should be controlled to avoid oxidative stress induced by high oxygen concentration. Both synergistic treatments should be combined with hemodynamic monitoring to avoid hyperventilation resulting in hypocapnia (exacerbation of pulmonary vasoconstriction) or oxygen therapy-associated lung injury and combined with PH-targeted drugs to reduce pulmonary vascular resistance in multiple ways. Ultimately, the strategy should be adjusted individually, considering protecting the right heart’s function and balancing systemic oxygen supply and demand to maximize the therapeutic effect.
With a deeper understanding of the pathogenesis of PH, genetically and molecularly targeted therapies have become a hot research topic.
Gene therapy corrects PH-related genetic defects by introducing normal genes or interfering with abnormal gene expression. Its preclinical studies have demonstrated clear therapeutic potential. Based on the mechanisms of action, existing research can be mainly divided into the following
In terms of inhibiting cell proliferation, studies have confirmed that Let-7a-transfected mesenchymal stem cells can restore BMPR2 expression by downregulating STAT3, inhibit the excessive proliferation of PASMCs, and thereby reverse established PH in rat models [147]. Prothymosin ɑ (ProT-ɑ) gene transfer inhibits the proliferation and fibrosis of PASMCs by upregulating ProT-ɑ, significantly reducing the mortality rate of model rats [148]. In the regulation of signaling pathways, injection of Sendai virus vector carrying caNPR2 into the left pulmonary artery can significantly increase local cGMP levels, inhibit PASMCs proliferation, and alleviate PVR without observing systemic side effects [149]. In vascular function regulation and endothelial-targeted therapy, in addition to adeno-associated virus (AAV)-mediated Apela or CTRP9 which can upregulate eNOS activity and inhibit vascular remodeling[150], endothelial overexpression of angiotensin-(1–7) driven by a hypoxia-responsive promoter can also effectively reverse hypoxia-induced PH[151]. Furthermore, AAV-mediated lung endothelium-specific GRK6 overexpression has also shown protective effects in reducing right ventricular systolic pressure and improving cardiopulmonary remodeling [152].
Molecularly targeted therapies focus on regulating core pathways induced by hypoxia drives metabolic reprogramming (Warburg effect) and mitochondrial fragmentation by stabilizing HIF-1ɑ; inhibitors targeting PDK (dichloroacetic acid) restore mitochondrial oxidative metabolism. Additionally, DRP-1 inhibitors (Mdivi-1) inhibit cell proliferation by blocking excessive mitochondrial fragmentation [153]. Hypoxia triggers epigenetic abnormalities (SOD2 gene silencing), which can be restored by HDAC inhibitors or DNA demethylating agents (5-azacytidine) to restore protective gene expression [154]. Furthermore, vasoactive intestinal peptide (VIP) analogs antagonize hypoxia-synergized inflammatory factors (IL-6 and TNF-ɑ) by antagonizing and repairing the BMPR2 signaling pathway, reversing PVR [155]. Besides, the inhibitors of the RhoA/ROCK pathway that inhibit the proliferation and migration of PASMCs and attenuate vascular remodeling, and targeted therapies against non-coding RNAs, including antisense oligonucleotides to inhibit aberrantly expressed miRNAs or IncRNAs, are expected to be a new strategy for PH treatment.
However, translating these preclinical achievements into clinical applications still faces multiple challenges. First, although animal models (such as chronic hypoxic rats or Sugen-hypoxia models) can simulate some PH phenotypes, their pathological mechanisms differ from those of human PH, especially in terms of the immune microenvironment and metabolic reprogramming. Second, the targeting ability and bioavailability of drug delivery systems in pulmonary blood vessels still need to be optimized. Furthermore, the high heterogeneity of PH requires individualized treatment strategies, yet there is a lack of reliable biomarkers to guide the selection of targeted therapies. In the future, multicenter clinical trials will be needed to verify the safety and efficacy of these novel therapies.
Multi-omics integration analyses provide new perspectives to investigate the molecular mechanisms of the hypoxic microenvironment in PH; however, they still face multiple challenges. At the data level, the heterogeneity and dynamics of different histological data make integration difficult [156]. The existing animal models can hardly accurately simulate the human hypoxic microenvironment. At the mechanistic level, the synergistic effects of the hypoxia signaling pathway with other PH-related pathways (metabolic reprogramming and inflammation) have not yet been elucidated [5]. There is limited validation of the causal relationship between molecular markers and phenotypes identified by multi-omics. At the clinical translational level, the heterogeneity of patients with PH and the lack of hypoxia-specific targeting strategies hinder the translation of histological results to diagnostic and therapeutic applications [157].
In the future, we need to focus on the following to achieve technological breakthroughs and develop single-cell/spatial multi-omics combined with dynamic network modeling tools to reveal the cellular heterogeneity and temporal pattern of hypoxia response. Optimization of models to construct three-dimensional organoids or gene editing models to simulate real microenvironments. Translational strategies to screen hypoxia-dependent biomarkers (metabolites or epigenetic targets) and explore co-targeted therapies. Interdisciplinary synergy, fusing computational biology and clinical resources to establish standardized analysis processes and multi-center cohorts to drive the development of precision intervention strategies.
AI technology has demonstrated significant potential for drug discovery and development. Using AI algorithms to analyze large amounts of biological data can accelerate the process of drug target discovery, drug design, and drug screening. In developing PH therapeutic drugs, AI can optimize the drug research and development (R&D) process, shorten the R&D cycle, and reduce R&D costs by predicting drug activity, toxicity, and pharmacokinetic properties. However, the application of AI technology in drug discovery and development is still in its early stage, facing core challenges including data scarcity (insufficient patient samples, differences between animal models and human pathology), microenvironmental complexity (multicellular interactions and cross-regulation of hypoxia signaling pathways), and barriers to clinical translation (insufficient model interpretability and patient heterogeneity).
In the future, it is imperative to build dynamic digital models by integrating multidimensional data (clinical-omics-imaging), mining hypoxia-related targets and existing drug reuse potential, and combining organoid, AI personalized prediction, and interdisciplinary collaboration platforms. This will facilitate the whole chain of innovations from mechanism analysis to precise treatment and ultimately break through the bottleneck of PH treatment.
The study of the hypoxic microenvironment in PH faces the dual challenge of gender and individualized differences. Sex differences are manifested by the paradoxical phenomenon of high prevalence in females and poor prognosis in males, which can be associated with the imbalance of sex hormones (bi-directional regulation of estrogens) and sex chromosome-associated genes [158]. However, the mechanism of their interaction with hypoxic signaling pathways has not yet been elucidated. Individual differences arise from genetic background heterogeneity, epigenetic modifications, and metabolic reprogramming, resulting in significantly different patient responses to the hypoxic microenvironment and therapeutic responses.
In the future, we need to break through the paradigm of “single-model” research and analyze the dynamic interactions between host characteristics and the microenvironment through gender-stratified experimental design (sex hormone regulation model) and multi-omics integration (genome, epigenome, and metabolome). Furthermore, we must develop precise therapeutic strategies (gender/genotype-directed targeted drugs and hypoxia-responsive smart drug delivery system), promote clinical research to incorporate individualized typing criteria (organoid models and AI efficacy prediction), and ultimately realize the transformation from population medicine to individualized medicine.
The hypoxic microenvironment plays a crucial role in PH development and involves a variety of complex cell biological and molecular biological mechanisms. Currently, some progress has been made in the clinical treatment of PH in the hypoxic microenvironment; however, multiple challenges exist. In the future, through the integration of multi-omics analysis, artificial intelligence-driven drug development, and focusing on gender and individual differences, it is expected that the pathogenesis of PH will be further unraveled. Furthermore, more effective therapeutic strategies will be developed to improve the prognosis and quality of life of patients with PH.