Pulmonary Vascular Signaling in Pulmonary Hypertension

Abstract

This review focuses on describing the potential pathogenic roles of endothelial Ca²⁺ and K⁺ signaling in the development and progression of pulmonary hypertension through its putative regulation of cellular senescence and inflammasome activation. Ca²⁺ influx through mechanosensitive and receptor-operated cation channels and Ca²⁺ release from the endoplasmic reticulum are involved in upregulating the cell cycle inhibitors p53, p21, and p16 (which result in cellular senescence) by activating the AKT/mTORC1 pathway in lung vascular endothelial cells (ECs). A rise in cytosolic Ca²⁺ concentration, resulting from Ca²⁺ influx and release in lung vascular ECs, is also necessary to activate both canonical (NLRP3) and non-canonical inflammasomes, thereby promoting vascular and perivascular inflammation. Furthermore, K⁺ efflux through multiple types of K⁺-permeable channels and pores (e.g., K⁺ ionophores, toxin-formed pores/channels, non-selective cation channels, and Ca²⁺-activated K⁺ channels) is sufficient for canonical (NLRP3) inflammasome activation. The senescent ECs release senescence-associated secretory phenotype (SASP) factors that subsequently cause endothelial-to-mesenchymal transition in adjacent ECs and promote cell proliferation/migration in adjacent smooth muscle cells and (myo)fibroblasts, leading to vascular remodeling and occlusive intimal lesions, and pulmonary hypertension.

Graphical Abstract

Pulmonary circulation is a low-resistance, low-pressure circulatory system that facilitates gas exchange. The deoxygenated venous blood is pumped from the right ventricle (RV) into the pulmonary artery, where it then travels through pulmonary arterioles to the lung capillaries, where CO₂ is released and O₂ is taken up during respiration. The oxygenated arterial blood in the pulmonary veins returns to the left atrium, which is then pumped from the left ventricle into the systemic arteries, reaching the entire body and all its organs. As part of the low-pressure system, the wall of the right ventricle is thin and cannot sustain a dramatic increase in afterload, for example, when pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) are increased in patients with pulmonary hypertension (PH). Indeed, RV failure is the common cause of death of patients with severe PH, and RV function is the primary determinant of the mortality of pulmonary vascular disease.

The lung vasculature is subjected to constant mechanical stimulation from airway radial traction and RV ejection or cardiac output (CO), as well as from shear stress at numerous bifurcations and junctions. Indeed, multiple mechanosensitive cation channels (MSCC), such as Piezo1 and TRPC6^1–3, and various mechanically-activatable membrane receptors, such as GPR68⁴, are functionally expressed in lung vascular cells and play important physiological and pathophysiological roles in regulating lung vascular function.

Pulmonary arterial pressure (PAP) is a function of CO and PVR: PAP = CO×PVR. PVR is positively proportional to the total length of the lung vessels (L) and the viscosity of the blood (η), and inversely proportional to the fourth power of the intraluminal radius (r) of the pulmonary artery: PVR = (8ηL)/(πr⁴). Therefore, a slight decrease in intraluminal diameter or radius would significantly increase PVR and PAP, and increase the afterload of the RV. Four pathophysiological and pathological changes in the lung vasculature directly contribute to the decrease in the intraluminal radius, thereby affecting PVR and PAP: sustained pulmonary vasoconstriction, concentric pulmonary vascular remodeling, in situ thrombosis and occlusive vascular lesions, and increased vascular wall stiffness.⁵

Cytosolic Ca²⁺ is a pivotal second messenger involved in a variety of cellular functions. An increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) is required for stimulating cell contraction and motility, cell proliferation and apoptosis, angiogenesis, and cell metabolism. Ca²⁺ signaling is tightly controlled by the function, expression, and activity of Ca²⁺-permeable cation channels and Ca²⁺ transporters in the plasma membrane and the endoplasmic (ER) and sarcoplasmic (SR) reticulum membranes. [Ca²⁺]_cyt is increased by Ca²⁺ influx through Ca²⁺-permeable cation channels in the plasma membrane and Ca²⁺ release from the SR/ER and other intracellular stores, whereas [Ca²⁺]_cyt is decreased by Ca²⁺ extrusion by the plasmalemmal Ca²⁺-Mg²⁺ ATPase (or Ca²⁺ pump) and Ca²⁺ sequestration into the ER by SR/ER Ca²⁺ pump (SERCA). When an increase in [Ca²⁺]_cyt occurs due to Ca²⁺ influx and/or Ca²⁺ release, it activates Ca²⁺-sensitive signaling proteins, such as calmodulin (CaM) and Ca²⁺/CaM-dependent kinases (CaMK), to regulate cell contraction and mobility, cell proliferation and division, cell metabolism, and inflammation. The cell-type-specific expression and cellular localization of Ca²⁺ signaling proteins control and fine-tune the downstream responses of Ca²⁺/CaM-signaling pathways involved in both physiological and pathophysiological processes across various cell types, such as vascular smooth muscle cells (SMCs) and endothelial cells (ECs). Upregulated Ca²⁺ channels and enhanced Ca²⁺ signaling in vascular SMC and EC are indeed implicated in the development and progression of various diseases, including pulmonary arterial hypertension (PAH)^6–8, systemic or essential hypertension, atherosclerosis, and stroke.

As an organ constantly under mechanical stress, the lungs and the lung vasculature express several mechanosensitive cation channels involved in Ca²⁺ signaling, which play essential roles in maintaining lung vascular function and in contributing to the pathophysiological and pathological development and progression of lung vascular disease. Since lung ECs are among the most abundant cell types in the lungs, here we focus on reviewing the roles of endothelial mechanosensitive cation channels (e.g., Piezo1 and TRPC6) in maintaining normal lung vascular EC function and their potential pathogenic roles in the development and progression of PH.

Distribution of Mechanosensitive Ion Channels in Various Lung Vascular Cells

Human lungs are composed, anatomically, of two lobes in the left lung (a superior or upper lobe and an inferior or lower lobe) and three lobes in the right lung (a superior or upper lobe, a middle lobe and an interior or lower lobe), while the mouse lungs consist of one lobe in the left lung and four lobes (a superior, a middle, an inferior and a post-caval lobe) in the right lung. Histologically, the lungs comprise airway, interstitial, and vascular tissues (including lymphatic vessel tissue). The lung vasculature consists of arteries, capillaries, and veins, with lung arteries and veins further categorized into large arteries and veins, medium-sized arteries and veins, small arteries and veins, and arterioles and muscular venules. The diameter of the human lung capillary is 4–10 μm (Fig. 1A). The lung arterial and venous vessels are structurally or histologically composed of three layers: the adventitia (mainly containing fibroblasts, myofibroblasts, and extracellular matrix), the media (primarily containing SMC), and the intima or the endothelium (mainly containing EC). Other cell types, such as vascular-resident progenitor cells, pericytes, and infiltrated immune cells, are found in the lung vascular wall and the perivascular niche of the lung parenchyma. All these cells contribute to the pathological vascular remodeling and occlusive lesions found in patients with pulmonary arterial hypertension (PAH) (Fig. 1B). When blood enters the pulmonary circulation system, the pressure drops by 25% in the arterial segment, 13% in the capillary, and 32% in the venous segment.⁵ These data indicate that functional and structural changes in arteries, capillaries, and veins all contribute to the regulation of pulmonary arterial pressure and pulmonary vascular resistance.

Figure 1.

Classification of the lung vascular cells and histological structure of the pulmonary vasculature. A: Lung angiography images of the pulmonary vasculature and diagram showing the major types of pulmonary vascular cells. B: Histological image (H&E staining) showing pulmonary artery (PA) with occlusive lesion and wall thickening, airway (A) and interstitial (Int) tissues in the lung of a patient with idiopathic pulmonary arterial hypertension. C: Schematic diagram showing the structure of artery (a), arteriole (b) and capillary (c), as well as the junction between the arteriole and capillary in which pericytes are shown (d). D: Elastic Van Gieson (EVG) staining of the lung tissue showing the cross section of a large pulmonary artery from a patient with pulmonary embolism in which the adventitia mainly containing fibroblasts (FB), the media (M) mainly consisting of smooth muscle cells (SMC), and the intima (I) or endothelium composed of endothelial cells (EC), as well as external and internal membranes are labeled. The arrows indicate fibrin in the emboli and its interaction with the intimal endothelial cells. (a) 10 times magnified image. (b) 60 times magnified image.

The early studies suggested that more than 50% of cells in the lungs are vascular and microvascular ECs.⁹ The single-cell RNAseq data in the LungMap database¹⁰ seem to indicate that the majority of cells in the lungs are type I epithelial cells, while vascular (mainly capillary) ECs make up about 30% of lung cells. There are multiple types or subtypes of cells that are histologically related to the pulmonary vasculature, composed of the conduit and resistance PA, arterioles, capillaries and veins: 1) endothelial cells (EC) including arterial and venous EC, capillary EC and other EC; 2) mesenchymal cells (MSC) including vascular SMC (VSMC), airway SMC, fibroblasts (FB), myofibroblasts (myoFB) and pericytes; and 3) immune cells (IM) infiltrated into or accumulated in the lung vasculature and parenchyma (Fig. 1C–D). A simple Western blot experiment indicates that EC markers, such as CD31, are the predominant proteins expressed in peripheral lung tissues. In contrast, SMC markers, such as SMA (or ACTA4), are the predominant proteins expressed in isolated pulmonary arteries.¹¹

Various ion channels and membrane receptors are expressed in the lung vasculature; however, the distribution of multiple channels and receptors, particularly those related to the development and progression of pulmonary vascular disease, can differ significantly among different subtypes of vascular cells. A good example of the differentiated distribution of the same channel/protein in lung vascular EC (or AEC) and SMC (or VSMC) is Piezo1/2 and TRPC6/V1 channels. In normal human lungs, Piezo1, a mechanosensitive cation channel, is highly expressed in all types of ECs. TRPC6, a receptor-operated cation channel sensitive to mechanical stimulation^2,12, is, however, highly expressed in vascular and airway SMCs. At the same time, TRPV1, the capsaicin receptor, seems to be highly expressed in all types of cells in the lung (Fig. 2A–C). By analyzing the expression ratio between arterial ECs and vascular SMCs (AEC/VSMC ratio), Figure 2D shows a list of genes (mRNAs) that are highly expressed in arterial ECs compared to their expression in vascular SMCs (i.e., with the highest ACE/VSMC ratio in expression), and a list of genes that are highly expressed in vascular SMCs in comparison to the expression level in arterial ECs (i.e., with the highest VSMC/ACE ratio). For example, Sox17, for which mutations or SNPs are indeed associated with PAH^13,14, is highly expressed in arterial ECs, along with TIE1, SLC6A4 (serotonin transporter), S1PR1 (sphingosine-1 phosphate receptor 1, a GPCR), Notch4 and TRPM6. In contrast, KCNA5, a gene encoding a voltage-gated K⁺ (Kv) channel pore-forming α subunit that has been shown to associate with hypoxia-induced PH and PAH^15–17, is highly expressed in vascular SMCs, along with KCNK17, Notch3, EDNRA (endothelin receptor A, a GPCR), and KCNK3 (a K⁺ channel gene implicated in PAH).^18,19 Among the selected mechanosensitive cation channels, acid-sensing ion channel 1–3 (ASIC1–3), as well as TRPC1, seem to be evenly distributed or expressed in arterial ECs and vascular SMCs. In contrast, Piezo2 (besides Piezo1), TRPC5 and TRPV4 channels are highly expressed in arterial ECs (and capillary ECs) in comparison to their expression level in vascular SMCs(Fig. 2E). The numbers for ASIC4 (AEC/VSMC, 0.00/0.10) and TRPA1 (0.00/0.07) are too small to be shown. The divergent or differential expression and distribution of various genes encoding ion channels and membrane receptors in vascular EC and SMC, which are adjacent to each other in the vasculature, indicate that their functional roles are specifically and uniquely defined in different types of cells.

Figure 2.

Cell types in the pulmonary vasculature and the distribution of ion channels among different lung cells. A: The pie chart of the proportion of different types of human lung vascular cells (excluding immune cells) based on the data in LungMAP¹⁰. B: UMAP visualization of major types of human lung cells, epithelial (Epi), endothelial (EC), mesenchymal (MSC), and immune cells (ImC) with different colors. Arterial endothelial cells (AEC), a subgroup of ECs, and vascular smooth muscle cells (VSMC), a subgroup of mesenchymal cells, are indicated by arrow lines. C: Differential expression of human PIEZO1, PIEZO2, TRPC6, and TRPV1 mRNA in distinct types of pulmonary cells (LungMAP) is shown, where high expression is depicted in red and low expression in blue. D: The ratio (AEC/VSMC) of the expression in arterial EC (AEC) and the expression in vascular SMC (VSMC) for selected genes. The higher the AEC/VSMC ratio is (bars in cyan), the higher the expression is in AEC (compared to VSMC). The lower the AEC/VSMC ratio is (bars in dark pink), the higher the expression in VSMC (compared to AEC). E: AEC/VSMC expression ratio of mechanosensitive channel genes. Note that TRPC6 is highly expressed in VSMC, while Piezo1 is highly expressed in AEC.

Mechanical Stress on Lung Vasculature and Mechanosensitive Cation Channels

The pulmonary circulation is a low-resistance and low-pressure system that enables the lungs to receive the entire cardiac output. The radial traction of airways and alveoli during respiration passively stretches the lung vasculature, playing a key role in maintaining low resistance and facilitating blood flow in the lungs. However, the RV ejection-associated pulmonary vascular expansion and the radial traction-mediated vascular stretch also subject the lung vasculature to consistent mechanical stimulation. Furthermore, the numerous bifurcations or branch junctions, as well as the short branches or segments of the lung arteries and arterioles (Fig. 3A), result in the lung vascular endothelium being subjected to consistent flow shear stress (Fig. 3A and B). Figure 3C depicts mechanical forces reinforced onto the lung vascular wall and vascular SMC/EC: shear stress, cyclic stretch, compression and tension, interstitial flow, hydrostatic and osmotic pressure.

Figure 3.

Mechanical stimulation that is commonly applied to the lung vasculature and lung vascular EC (LVECs). A. Human pulmonary artery cast (a) and mouse pulmonary angiography images (b) showing numerous bifurcations or branch junctions in the lung vasculature. B: Blood flow (a) through the pulmonary artery (PA) causes the flow shear stress (especially in the bifurcations); pulmonary arterial pressure (PAP, a function of cardiac output and vascular resistance) reinforces the pressure onto the vascular wall and LVECs; and cardiac output (and airway radial traction) mediates vascular expansion and causes the stretch of vessel wall and LVECs (b). C: Schematic diagram showing different mechanical stimuli to the lung vasculature and LVECs: shear stress (a), cyclic stretch (b), compression (c), interstitial flow-mediated impact onto vessel wall (d), hydrostatic and osmotic pressure-mediated membrane stretch for vascular cells, including LVECs (e), and tension due to vasoconstriction (f).

PH is classified into five groups: i) PAH (Group 1), ii) PH associated with left heart disease (Group 2), iii) PH associated with lung diseases and/or hypoxia (Group 3), iv) PH associated with pulmonary artery obstructions including chronic thromboembolic PH (CTEPH), and v) PH with unclear and/or multifactorial mechanisms (Group 5).²⁰ PAH, hypoxia-mediated PH, and CTEPH are also defined as precapillary PH, whereas PH associated with left heart disease is considered postcapillary PH. Increased PAP in patients with PH or in animals with experimental PH increases vessel wall stress and strain (Fig. 3A) and may enhance endothelial fluid shear stress at the numerous bifurcations and junctions of the lung vasculature.²¹

Laminar flow is shown to induce dilative and protective (e.g., anti-inflammatory) effects on systemic arteries. In contrast, turbulent flow in blood vessel bifurcations is associated with the formation of obliterative thromboemboli and the development of atherosclerotic lesions in the systemic circulation.²² In the pulmonary circulation, there are numerous branches and junctions in the pulmonary vasculature (as shown in Figs 1A and 3A). The lungs are also the only organ that receives the entire cardiac output. In addition to inducing endothelial NO release and endothelium-dependent vasodilation, the flow shear stress in distal arteries may contribute to the development of PH by releasing endothelium-derived constricting factors, inflammatory cytokines, and mitogenic factors. Wall shear stress (WSS) is positively proportional to blood-flow velocity and blood viscosity (μ) and inversely proportional to the intraluminal radius of blood vessels: WSS = (4μQ)/(πr³), where Q is blood flow rate and r is the intraluminal radius of blood vessel.²³ Sustained vasoconstriction, concentric vascular remodeling (or wall-thickening), in situ thrombosis, and occlusive intimal lesions are the major causes of elevated PVR and, subsequently, increased PAP. In all pre-capillary PH (i.e., Group 1/PAH, Group 3/PH with respiratory diseases and HPH, Group 3/CTEPH), the intraluminal diameter in distal pulmonary arteries decreases due to concentric thickening of the PA wall, obliterative vascular lesions (e.g., neointimal and plexiform lesions), and sustained pulmonary vasoconstriction. Therefore, flow shear stress is increased in the precapillary PH groups and implicated in the development and progression of pulmonary vascular remodeling.^21,24,25 In CTEPH associated small vessel disease (or CTEPH with persistent postoperative PH), the partially or completely occluded distal arteries by emboli or fibrotic emboli result in the redirection or redistribution of blood flow to adjacent arteries, which may increase flow shear stress as a pathogenic mechanism for distal arterial remodeling.^26,27 PH associated with hypoxia (e.g., high-altitude PH), increased blood viscosity further increases the shear stress on the lung vasculature. Nevertheless, the computational and tomographic analyses of pulmonary hemodynamics indicate that the wall shear stress in proximal arteries is decreased in patients with severe PH (e.g., in patients with PAH),^23,28–30, which is potentially due to decreased cardiac output and right heart failure. One focus of this review is to discuss the potential role of mechanosensitive Ca²⁺-permeable cation channels and enhanced Ca²⁺ signaling in lung vascular ECs in the development and progression of vascular remodeling in pulmonary vascular disease.

Multiple mechanosensitive channels and receptors are expressed in lung vascular cells, including FB/myoFB, SMC, and EC. In addition to mechanically activated channels (Fig. 2E), mechanosensitive receptors, such as GPR68^4,31, Src³², angiotensin I type 1 receptor (AT1R), histamine H1 receptor (H1R), vasopressin V1A receptor and adhesion GPCR are expressed in vascular cells (EC and SMC). These mechanosensitive channels and receptors enable cells (e.g., lung vascular EC) to respond to physical forces in their local environment (e.g., flow shear stress, compression and tension, pressure and stretch) and transduce these mechanical signals into intracellular modification of gene transcription and expression, enzyme activity and substrate catalysis, protein trafficking and distribution and, ultimately, contributing to the precise regulation of cellular function and homeostasis. Here, we focus on reviewing how mechanosensitive Ca²⁺ signaling via MSCC regulates cellular senescence and inflammation, and on their potential role in the development of pulmonary vascular disease.

Ca²⁺ signaling in pulmonary artery smooth muscle cells (PASMC) and other types of mesenchymal cells (e.g., fibroblasts, myofibroblasts, and pericytes) in the lungs plays an essential role in the development and progression of PAH/PH. An increase in [Ca²⁺]_cyt due to Ca²⁺ influx through plasmalemmal cation channels and Ca²⁺ release from the intracellular stores (mainly the sarcoplasmic reticulum) is a major trigger for vasoconstriction. Ca²⁺ binds to or activates calmodulin (CaM), and Ca²⁺/CaM then activates myosin light chain kinase (MLCK), which leads to the phosphorylation of myosin light chain (MLC) and causes PASMC contraction and pulmonary vasoconstriction. Increases in cytosolic, intracellularly stored, and nuclear [Ca²⁺] are also necessary stimuli that help propel cells through the cell cycle, thereby promoting cell proliferation and contributing to pulmonary arterial wall thickening and muscularization of arterioles and capillaries. The Ca²⁺-sensitive signaling proteins and transcription factors are involved in activating at least four steps in the cell cycle: the transition from G₀ to G₁, the transition from G₁ to DNA synthesis phase (S), the transition from G₂ to M, and mitosis (M) itself. The Ca²⁺ or Ca²⁺/CaM/CaMK-associated cell proliferation in PASMC, lung (myo)fibroblasts, and pericytes also contributes to the muscularization of arterioles and capillaries, contraction and remodeling of capillaries surrounded by pericytes,³³ and formation of occlusive intimal lesions (e.g., neoplasm and plexiform lesions) in PAH.

The role of Ca²⁺ signaling and cation channels in PASMCs and other types of mesenchymal cells in PH is discussed extensively in other recent reviews.^5,34 This review focuses on the pathogenic role of Ca²⁺ signaling in driving (or linking) pathological vascular remodeling in PH by inducing cellular senescence and inflammasome activation in lung vascular endothelial cells.

Role of Ion Channels and Ca²⁺ Signaling in Cellular Senescence

Cellular senescence, a specific cell state distinct from G₀ or a terminally differentiated state, can occur in various cell types.^35,36 Based on the inducers, cellular senescence can be classified into replicative senescence, oncogene-induced senescence, therapy-induced senescence, mitochondrial dysfunction-induced senescence, and immunologically induced senescence.³⁷ The cell cycle of the senescent cell is stably arrested, which is mediated by the activation of two cyclin-dependent kinase (CDK) inhibitors, p21^WAF1/Cip1 and p16^INK4A, as well as a tumor suppressor, p53 (which increases p21). The p21- and p16-mediated inhibition of CDK4/cyclin D and CDK2/cyclin E, respectively, leads to the cell cycle arrest in G₁ phase.³⁷ Besides the permanent cell cycle arrest, senescent cells acquire a secretory character, the senescence-associated secretory phenotype (SASP). The secreted substances from senescent cells, also termed as SASP factors, include pro-inflammatory cytokines and chemokines (e.g., IL-6, TNF-α, CCL-2), growth factors (e.g., PDGF-AA, TGF-β), bioactive lipids (e.g., ceramide, prostaglandins, leukotriens), and extracellular matrix (ECM)-associated proteases (e.g., matrix metalloproteinases, ADAM17), which cause pro-inflammatory, pro-fibrotic, and proliferative effects on adjacent cells via a paracrine mechanism.³⁸ The SASPs released from primary senescent cells also induce secondary senescence (or paracrine senescence) in neighboring cells, promoting the progression of cellular senescence in the endothelium. The mitogenic and fibrotic SASPs released from primary and secondary senescent cells promote the expansion and progression of cell proliferation and tissue fibrosis in the local environment, such as the perivascular niche in the lungs and the obliterative thromboemboli in small pulmonary arteries and arterioles (Fig. 4). Cellular senescence can be induced by various stress stimulus, including DNA damage, oncogene activation, oxidative stress, and genotoxic agents.^39–41

Figure 4.

Ca²⁺ signaling through Ca²⁺ influx contributes to inducing lung vascular EC senescence and vascular endothelial dysfunction and damage. A: Excessive Ca²⁺ influx through mechanosensitive cation channels (e.g., TRPC6, Piezo1) and receptor-operated cation channels (e.g., TRPC6, TRPV1) is involved in inducing cellular senescence by increasing p53, p21 and p16 through an AKT/mTOR signaling pathway in LVECs. Cellular senescence in LVECs subsequently results in the decrease of nitric oxide (NO) and other endothelium-dependent relaxing factors (EDRF). The EC senescence-mediated release of the senescence-associated secretory phenotype (SASP) factors (e.g., IL-6, TGF-β, mtDNA) can induce vascular inflammation and lead to secondary senescence in adjacent LVECs and other vascular cells, ultimately contributing to pulmonary vascular remodeling and the development of PAH/PH. B: Pulmonary vascular remodeling is shown as the reduced number of lung vascular branches and junctions in pulmonary hypertension (PH) mice (ex vivo angiogram, upper panels) and the increased PA wall thickening and occlusive lesions in patient with pulmonary arterial hypertension (PAH) (H&E staining of lung tissues, lower panels), in comparison to the normal controls (Nor).

For the lung vascular endothelium, hemodynamic stress, mechanical stretch, as well as hyperoxia and hypoxia, can all serve as stimuli to induce senescence through mechanosensitive signaling. Lung vascular ECs form a single layer of cells that line all segments of the pulmonary vasculature, including the proximal (i.e., conduit and large arteries), distal (i.e., small arteries and arterioles), and capillary vessels. Therefore, lung vascular ECs encounter not only various metabolites and factors in the blood but also the flow shear stress induced by pulmonary blood flow and vascular wall stretch induced by airway radial traction and RV ejection-mediated vascular extension and recruitment (Fig. 3). While high laminar shear stress is shown to help maintain EC function in the systemic circulatory system, low and oscillatory shear stress, such as those in the pulmonary circulation, cause ECs to obtain features similar to SASP and senesence.^42–44 Furthermore, hyperoxia can promote cellular senescence.^45–47 The pulmonary vasculature is exposed to a higher partial pressure of oxygen (PO₂) from surrounding alveoli (alveolar PO₂ is 100 mmHg) than systemic vessels in various organs/tissues. In comparison, lung capillaries and postcapillary venules also encounter high arterial PO₂ (100 mmHg under normal conditions), making pulmonary ECs more vulnerable to hyperoxia- and/or ROS-mediated cellular senescence than ECs in other systemic vascular beds.

Senescent EC has reduced NO production, increased expression of inflammatory cytokines, and SASP factor secretion, resulting in inhibited endothelium-dependent vasodilation, reduced angiogenic ability^48–50, and impaired barrier function.⁵¹ Due to the functional changes, senescent EC has been shown to play an important pathogenic role in the development and progression of cardiopulmonary and vascular diseases, such as PAH, acute and chronic lung injury, atherosclerosis, heart failure, chronic obstructive pulmonary disease (COPD)^52,53, and Kawasaki disease.^54–56 PAH is a fatal disease characterized pathologically by concentric vascular remodeling and occlusive intimal lesions, which increase pulmonary vascular resistance and pulmonary arterial pressure.^57–59 Endothelial dysfunction and injury have been demonstrated to play a crucial pathogenic role in the development of PAH,⁶⁰ while cellular senescence in lung vascular EC is implicated in the development and progression of PH/PAH. First, senescent EC is observed in the remodeled lung vessels of PAH patients.^61,62 The main regulators of cellular senescence, p53, p21 and p16 are upregulated in whole-lung tissues and pulmonary vascular EC in patients with Group-1 PH (PAH) and Group-3 PH (PH associated with lung diseases and/or hypoxia).^35,62,63 Senescent EC has also been observed in animals with experimental PH (e.g., hypoxia-induced PH and monocrotaline-induced PH),^35,62,64 suggesting that cellular senescence in lung EC is a common phenomenon among different groups of PH. Enhanced lung vascular remodeling in association with the increased severity of PH was observed in EC-specific progeroid mice with experimental PH.⁶² Knockdown of frataxin (a mitochondrial protein) promotes EC senescence, and EC-specific knockout of frataxin exacerbates hypoxia-induced PH in mice.³⁵

Nestin, which is regulated by SOX17, suppresses EC senescence; while overexpression of SOX17 and knockdown of Nestin ameliorate MCT-induced PH in rats.⁶³ Furthermore, senolytic agents that specifically kill senescent cells have been shown to reverse PH in animal models.³⁵ Hypoxia has been shown to induce EC senescence in vitro and in animals (mice) with hypoxia-induced PH. Other pathogenic causes for PH, including frataxin deficiency,³⁵ IL-11 and soluble IL-11Rα (which is upregulated in PH patients)⁶⁵, and SOX17 deficiency⁶³ all have been demonstrated to induce lung vascular EC senescence. One of the current therapeutic targets for PAH is the endothelin pathway; endothelin receptor antagonists (ERAs), such as bosentan and ambrisentan, have been used to treat different forms of PH^66–69. Endothelin-1 induces cellular senescence, while senescent cells can produce and release high level of endothelin-1⁷⁰. Increased endothelin-1 is also shown to be the trigger for hyperphosphatemia-associated cellular senescence in EC; blockade of endothelin receptors with bosentan inhibits EC senescence in hyperphosphatemia⁷¹. Another current PAH drug target, Activin A, which belongs to the TGF-β superfamily, has been identified as a SASP.⁷². These studies strongly suggest an important pathogenic role of EC senescence associated with lung endothelial dysfunction and injury in PAH/PH; while inhibition of cellular senescence in EC or SASP release from senescent cells is involved in the therapeutic effects of ERAs and TGF-β inhibitors for PAH/PH.

Intracellular Ca²⁺, or an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt), functions as a second messenger to activate a variety of intracellular signaling pathways. Ca²⁺ signaling, resulting from spatiotemporal Ca²⁺ influx and release, is shown to play a pivotal role in the induction and regulation of cellular senescence.^73,74 Elevated intracellular Ca²⁺ in the cytosol and mitochondria due to Ca²⁺ influx through Ca²⁺-permeable cation channels in the plasma membrane and Ca²⁺ release through inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) in the endoplasmic reticulum (ER) has been observed and implicated in senescent cells^73,74, whereas removal (or chelation) of extracellular and intracellular free Ca²⁺ with BAPTA (a potent chelator of Ca²⁺) inhibits and prevents cellular senescence, by inhibiting DNA damage response and p53/p21 pathway activation. These observations suggest that an increase in [Ca²⁺]_cyt triggers cellular senescence. Several Ca²⁺- or Ca²⁺/calmodulin (CaM)-sensitive signaling pathways are involved in Ca²⁺-induced cellular senescence. A Ca²⁺/CaM/calcineurin/NFAT axis is indicated to upregulate calbindin, a Ca²⁺ binding protein, which is engaged in buffering the rise in [Ca²⁺]_cyt in senescent ECs.⁷⁵ An increase in AKT phosphorylation or activation, which can be induced by receptor-mediated PI3K/Ca²⁺/AKT signaling axis, is involved in inducing cellular senescence via p53 and p21.⁷⁶ AKT-mediated activation of mTORC1 stabilizes p21 by increasing 4E-BP1 without activation of p53. The AKT/mTORC1/4E-BP1-mediated increase in p21 inhibits the cell cycle⁷⁷ and induces cellular senescence. Under physiological conditions or at the physiological level, AKT signaling leads to the activation of MDM2, an E3 ligase that promotes the degradation of p53, thereby maintaining a low p53 level. Chronic hyperactivation or constitutive activation of AKT, such as when sustained Ca²⁺ influx (and release) occurs upon receptor activation and mechanical stimulation, results in mTORC1-dependent increases in p53 translation. The resultant activation or increase of p53/p21 contributes to cellular senescence by inhibiting CDK4/6 and cyclin D.⁷⁸ Cooperation between AKT and p21 has also been shown to result from p53-mediated activation of mTORC2, an upstream stimulator of AKT. In this scenario, p53 not only directly stimulates p21 but also activates mTORC2/AKT, leading to an increase in ROS via Nox4⁷⁹. Increased intracellular Ca²⁺ is also involved in downregulating miRNAs (e.g., miRNA-125 b, 504) that can directly bind to p53, increasing its mRNA degradation and inhibiting its translation.^80,81

Piezo1 is a mechanosensitive, non-selective cation channel with high permeability to K⁺, Na⁺, and Ca²⁺, while TRPC6 is a receptor-operated cation channel that is sensitive to mechanical stimulation.^1,11,82 It has been demonstrated that the expression of Piezo1 is upregulated in senescent chondrocytes, while activation of Piezo1 promotes cellular senescence in chondrocytes and the chelation of intracellular Ca²⁺ can inhibit the Piezo1-associated senescence.⁸³ Piezo1, along with Piezo2, is expressed in lung vascular ECs; the expression level of Piezo1 in lung arterial ECs is six times greater than that in lung vascular SMCs (Fig. 2E). Our previous study shows that Piezo1 is upregulated in lung vascular ECs and SMCs from patients with PAH and animals with experimental PH.^11,84 Hypoxia upregulates Piezo1 in pulmonary vascular EC and SMC. Piezo1 is upregulated in senescent chondrocytes and aged cartilage tissue, indicating that Piezo1 is responsible for increased [Ca²⁺]_cyt in chondrocyte senescence. Since hypoxia is also a cellular senescence stimulator, whether Piezo1, by mediating mechanosensitive Ca²⁺ entry, plays a pathogenic role in PH by regulating EC senescence warrants further investigation.

TRPC channels are receptor-operated, non-selective cation channels that allow Na⁺ and Ca²⁺ to go through. Upon ligand-mediated activation of G protein-coupled receptors (GPCRs) or tyrosine kinase receptors (TKR), the second messenger diacylglycerol (DAG) activates TRPC channels (e.g., TRPC6) and induces receptor-operated Ca²⁺ entry (ROCE), and IP₃ activates IP₃ receptor, a Ca²⁺ release channel, in the ER membrane, inducing Ca²⁺ release. When intracellularly stored Ca²⁺ in the ER/SR is depleted or actively depleted by IP₃-mediated Ca²⁺ mobilization through IP₃ receptors, STIM1 translocates to the puncta, recruits Orai1/2 channels in the plasma membrane to form store-operated Ca²⁺ channels (SOCC) and induces store-operated Ca²⁺ entry (SOCE). Lung vascular ECs and SMCs functionally express all the components for ROCE (e.g., TRPC channels) and SOCE (e.g., STIM/Orai); however, the voltage-dependent Ca²⁺ channel expression level in lung vascular ECs is extremely low, while its expression in pulmonary arterial SMCs is high. TRPC7, a ROCC closely related to TRPC3 and TRPC6, which are activated directly by DAG, has been shown to contribute to radiation-induced Ca²⁺ influx and ROS increase, likely leading to increased cellular senescence.⁸⁵ In addition, blockade of L-type voltage-dependent Ca²⁺ channels (VDCC) with isradipine inhibits PI3K/AKT and p53/p21-dependent cellular senescence in human neuroblastoma cells induced by mitochondrial complex I inhibitor (rotenone).⁸⁶ Furthermore, IP₃R2, a Ca²⁺ release channel expressed in the ER/SR, and TRPC3, a ROCC expressed in the plasma membrane and the ER/SR, have been shown to regulate cellular senescence by modulating Ca²⁺ transportation from the ER/SR to mitochondria^87–89, thus leading to ROS production and mitochondrial dysfunction. These studies all indicate a significant role of Ca²⁺ signaling in regulating cellular senescence.

DNA replication stress inducers and DNA-damaging agents, or genotoxic agents, are common inducers of cellular senescence, such as the DNA topoisomerase inhibitors, doxorubicin (an inhibitor of topoisomerase II) and mitoxantrone. Fragmented DNA in the cytosol resulting from nuclear DNA damage and fragmentation and mitochondrial DNA (mtDNA) release are implicated in activating intracellular Ca²⁺ signaling pathways involving cGMP, cGAS (cGMP-AMP synthase) and STING.^90,91 Single-stranded RNA has been shown to activate mechanosensitive cation channels such as Piezo1⁹² and increase [Ca²⁺]_cyt. Plasma cell-free DNA levels are associated with disease severity and survival in pulmonary arterial hypertension.⁹³ It is interesting to investigate whether extracellular DNA and RNA, along with cytosolic DNA/RNA can directly activate the plasmalemmal cation channels and induce Ca²⁺ influx and contribute to cellular senescence in lung vascular ECs and SMCs.

Role of Ion Channels and Ca²⁺ Signaling in Activation of Inflammasomes

Vascular and perivascular inflammation is implicated as one of the major causes for concentric vascular remodeling and occlusive intimal lesions in PAH/PH.^94–99 Infiltrated circulating immune cells (e.g., macrophages and neutrophils) in the pulmonary vascular wall provide mitogenic and fibrotic factors that stimulate proliferation of SMC and (myo)fibroblasts in the vascular wall, contributing to concentric vascular remodeling in PH. Infiltrated immune cells during vascular inflammation can also release cytokines/chemokines and growth factors that lead to the phenotypic transitions of vascular cells (e.g., endothelial-to-mesenchymal transition, EndMT, in EC and contractile-to-proliferative phenotypic transition in SMC). Furthermore, pro-inflammatory and pro-fibrotic factors (e.g., IL-1β/IL-18, HMGB-1) released from non-immune cells (e.g., vascular ECs), in which noncanonical and canonical (NLRP3) inflammasomes are activated by distinct mechanisms, play an important pathogenic role in the development of vascular remodeling and intimal lesions contributing to PAH/PH.

Inflammasomes are cytosolic multiprotein complexes that form in response to pathogen-associated molecular patterns (PAMPs) from pathogens or damage-associated molecular patterns (DAMPs) released by damaged cells. Inflammasome activation is a crucial process in innate immunity. Most inflammasomes are composed of three parts: the pattern-recognition receptors (PRRs) that recognize specific PAMPs or DAMPs, the adapter protein ASC, and the effector caspase (e.g., caspase-1). The inflammasome thus serves as a platform for caspase-1 activation.¹⁰⁰ Two steps are required for inflammasome activation: priming and activation. Not all the components of the inflammasome are constitutively expressed. Some of the components are only expressed at a high level after inflammatory stimulation. Hence, the activation of the inflammasome is typically a two-step process that facilitates the tight regulation of this pivotal immune response. During the first priming step, stimuli (e.g., lipopolysaccharide, LPS) recognized by other pattern recognition receptors (e.g., TLR4) induce the expression of inflammasome-related genes (e.g., NLRP3 and IL1B) by activating the transcription factor NF-κB. Moreover, the priming step also induces posttranslational modification of NLRP3, making it ready for the second activation or formation step.¹⁰¹ There are multiple stimuli for the second activating process, such as extracellular ATP and bacterial pore-forming toxins (e.g., antinoporins). When specific inflammasome PRRs recognize the second activating stimuli, the PPRs oligomerize and recruit ASC. The recruited ASC then oligomerizes, which further recruits pro-caspase-1. Due to the proximity, the recruited pro-caspase-1 autoactivates itself and auto-processes into caspase-1. Activated caspase-1 then processes pro-IL-1β and pro-IL-18 into their mature forms. Activated caspase-1 also processes gasdermin D (GSDMD). After processing, the N-terminus of GSDMD is released and inserted into the cell surface membrane, forming channels or pores. The mature IL-1β and IL-18 can be released through the GSDMD channel/pore. The GSDMD pore ultimately induces cell lysis due to the osmotic stress and results in pyroptosis, a form of lytic cell death (Fig. 5). Inflammasomes are named after their PRRs. Different PRRs have their own specificity to different stimuli. Among the various inflammasomes, the NLRP3 inflammasome is unique from other types of inflammasomes due to its versatile activators, which enables its participation in different infectious and sterile inflammatory diseases.¹⁰¹ Although NLRP3 inflammasome forms mainly in immune cells, other cell types, including ECs, have also been shown to have NLRP3 inflammasome activation under stress.¹⁰²

Figure 5.

Ca²⁺ influx through receptor-operated and mechanosensitive cation channels (MSCC) and K⁺ efflux through Ca²⁺-activated K⁺ channels (KCa-Ch) contribute to inflammasome activation in lung vascular endothelial cells (ECs). Inflammasome activation is a two-step process: the priming step involves the transcriptional upregulation of inflammasome proteins (e.g., NLRP3, pro-IL-1β and pro-caspase-1), followed by the activation step that requires assembly of the inflammasome complex, activation of caspase-1 and release of inflammatory cytokines IL-1β/IL-18 through N-terminal GSDMD-formed pores/channels in the plasma membrane. A variety of endogenous and exogenous stimuli can stimulate noncanonical (caspase-4/5 or caspase-11 in mice) and canonical NLRP3 (caspase-1) inflammasome activation, and lead to pyroptotic cell death. During the priming step, the NF-κB-mediated upregulation of inflammasome components may be associated with upregulation of ion channels (e.g., Piezo1, TRPC6 and KCNN2) and membrane receptors (e.g., CaSR) to ensure the necessary Ca²⁺ influx and K⁺ efflux. The excessive Ca²⁺ influx and K⁺ efflux then regulate both the noncanonical (caspase-4/5) and canonical (NLRP3, caspase-1) inflammasome activation. The ultimate release of IL-1β/18 and pyroptotic EC death results in (peri)vascular inflammation and vascular remodeling that contribute to the development of pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH).

In addition to the canonical inflammasomes, such as NLRP3 inflammasomes (caspase-1), there are non-canonical inflammasomes whose formation results in the activation of caspase-4 (in humans)^103–106 or −11 (in mice) and leads to the formation of GSDMD channels/pores and release of IL-18/1β. Cytosolic LPS (an endotoxin) resulting from bacterial infection and LPS endocytosis can directly bind to caspase-4/5 and are reported to trigger non-canonical inflammasome activation.¹⁰⁴ LPS can bind to caspase-4/5 or caspase-11, forming an LPS-caspase-4/5 complex (or an LPS-caspase-11 complex) that facilitates the oligomerization of caspase-4/5 (or caspase-11) and results in the formation of a non-canonical caspase-4/5 (or caspase-11) inflammasome. The subsequent activation or cleavage of caspase-4/5 (or caspase-11) cleaves the N-terminus of gasdermin D (GDSMD), promoting the formation of N-GDSMD channels/pores in the plasma membrane.^107–109 The increased K⁺ efflux (and/or Ca²⁺ influx) through the caspase-4/5 (or caspase-11)-activated N-GDSMD channels/pores (and/or other non-selective cation channels) can stimulate or facilitate canonical NLRP3 inflammasome activation.^110,111

Well-controlled canonical (NLRP3/caspase-1) or non-canonical (LPS/caspase-4/5) inflammasome activation in immune cells has beneficial effects on controlling infection and eliminating damaged cells. In contrast, uncontrolled inflammasome activation is associated with various acute (e.g., sepsis) or chronic (e.g., atherosclerosis) inflammatory diseases. As the lining of blood vessels, EC dysfunction is involved in multiple vascular diseases. Activation of canonical (NLRP3/caspase-1) and/or non-canonical (LPS-caspase-4/5) inflammasomes in vascular EC induces the release of IL-1β and IL-18, which can further increase vascular inflammation and upregulate adhesion molecules expression, leading to leukocyte adhesion, occlusive intimal lesion and vascular remodeling. The resultant formation of N-GDSMD channels/pores, followed by pyroptosis in vascular EC, also results in endothelial dysfunction/damage and inhibits endothelium-dependent vasodilation and angiogenesis. Perivascular and vascular inflammation, as well as inhibited endothelium-dependent vasodilation, are important pathogenic causes of PAH and PH. Similar to IL-1β/IL-18, HMGB1 can be released during NLRP3 inflammasome activation in lung ECs to induce lung barrier dysfunction via reorganization of intracellular actin fibers and disruption of cell-cell junction cadherins.¹¹² Besides the release of inflammatory cytokines, pyroptosis induced by NLRP3 inflammasome activation results in loss of endothelial barrier function in lung injury, hence the neutrophil infiltration and lung edema.^113,114 It has been shown that the serum or plasma levels of IL-1β and IL-18 are higher in PAH patients as compared to normal controls.^115–117 The serum HMGB1 level is also higher in PAH patients than in healthy subjects.¹¹⁸ Activation of the NLRP3 inflammasome, demonstrated by expression of matured IL-1β, IL-18 and caspase-1 and higher NLRP3 expression in the lung tissues, is observed in animals with experimental PH, such as mice with hypoxia-induced PH and rats with MCT-treated PH.^119–121 Inflammasome-associated pyroptosis in EC has also been shown to play a significant role in the development of PAH/PH.¹²² Inhibition of inflammasome activation and pulmonary EC pyroptosis by DYZY01 restrains the development and progression of experimental PH.¹²³ Pyroptosis also causes the release of DAMPs, including ATP, which further exacerbates inflammation. Knockout of the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) in inflammasome activation and knockout of IL-1R both ameliorate experimental PH in animal models.^119,124 Moreover, BMPR2 deficiency promotes NLRP3/gasdermin E (GSDME)-mediated endothelial cell pyroptosis in PAH.¹²⁵

P2RX7 is a purinergic receptor that functions as a ligand-gated cation channel. Extracellular ATP, as a ligand for P2RX7, as well as P2RX7 function and expression are implicated in the pathogenic mechanisms of PH/PAH.^126,127 Increased expression level of P2RX7, along with increased NLRP3 and caspase-1, is demonstrated in the lung tissues of animals with experimental PH or rats with MCT-induced PH, while treatment of PH animals with the antagonist of P2RX7 inhibits NLRP3 inflammasome activation and attenuates RV systolic pressure and RV hypertrophy in rats with MCT-induced PH.¹²⁸ Pirfenidone (PFD), an anti-fibrotic drug that inhibits NLRP3 inflammasome activation and inflammation, has been shown to repress IL-1β and IL-18 maturation and ameliorates PH in rats with experimental PH.¹²¹ The IL-1R antagonists, such as anakinra, have also been reported to ameliorate hypoxia-induced PH in mice and MCT-induced PH in rats.^124,129 In patients with PAH, anakinra treatment lowers high-sensitivity C-reactive protein (hsCRP) levels and improves the symptoms of RV failure.¹³⁰ Furthermore, inhibition of the ET-1/ET_A signaling and elevation of cytoplasmic cGMP are both therapeutic strategies for developing novel treatment for PAH. ET-1 activates the NLRP3 inflammasome via Ca²⁺-mediated ROS production.¹³¹ Praliciguat, a stimulator of the soluble guanylate cyclase (sGC), and TPN171H and Avanafil, inhibitors of phosphodiesterase type 5 (PDE-5), all inhibit the NLRP3 inflammasome inhibit NLRP3 inflammasome activation^132–134 and ameliorate experimental PH in different animal models. These studies further strengthen the involvement of inflammasome activation in the development and progression of PAH/PH, while inhibition of inflammasome activation (in different types of cells) may be an important therapeutic strategy for developing new treatment for PH/PAH associated with (peri)vascular inflammation.

Why and how the NLRP3 inflammasome is activated by versatile stimuli is not fully understood. Among the mechanisms that have been suggested, the induction of ion flux, more specifically Ca²⁺ influx and K⁺ efflux (Fig. 5), is a common event for various NLRP3 inflammasome activators.^101,135 Ca²⁺ influx, along with Ca²⁺ release from the intracellular stores, contributes to NLRP3 inflammasome activation. One of the significant inflammasome stimulators activation is extracellular ATP, which can activate both P2X and P2Y receptors in the plasma membrane. P2X receptors are ligand-gated ion channels that are highly permeable to Ca²⁺ ions¹³⁶ and P2Y receptors are GPCRs that are functionally coupled to receptor-operated Ca²⁺ channels via IP₃ and DAG. ATP-mediated activation of P2X receptors (e.g., P2RX7) directly causes Ca²⁺ influx, while activation of P2Y channels (e.g., P2Y6)¹³⁷ indirectly causes Ca²⁺ influx and release by increasing DAG and IP₃. DAG activates receptor-operated cation channels (e.g., TRPC6 channels) to induce Ca²⁺ influx or receptor-operated Ca²⁺ entry, and IP₃ activates the IP₃ receptors or Ca²⁺ release channels in the ER (or SR) membrane to induce Ca²⁺ release or mobilization. Furthermore, the IP₃-mediated active depletion of Ca²⁺ from the ER/SR induces store-operated Ca²⁺ entry through STIM/Orai channels. The pore-forming toxins and the complement membrane attack complex (MAC), which are also inflammasome activators, can induce a massive influx of Ca²⁺. The Ca²⁺-induced Ca²⁺ release through ryanodine receptors is also implicated in inflammasome activation.¹³⁸ More importantly, the chelation of extracellular Ca²⁺ with EGTA (a Ca²⁺ chelator) and intracellular Ca²⁺ with BAPTA-AM (which is a membrane-permeable BAPTA, a stronger Ca²⁺ chelator than EGTA) can significantly inhibit NLRP3 inflammasome activation and IL-1β secretion in multiple cell types.^138–140 The data seem to indicate that Ca²⁺ influx and/or release is necessary, but may not be sufficient, to promote NLRP3 inflammasome activation. It is still unclear how intracellular Ca²⁺, or an increase in [Ca²⁺]_cyt through Ca²⁺ influx and/or release, causes or promotes inflammasome activation. Two potential mechanisms related to the role of Ca²⁺ in NLRP3 inflammasome activation are that a) a rise in local cytosolic Ca²⁺ may enhance NLRP3 and ASC interaction and b) an excessive increase in cytosolic Ca²⁺ due to Ca²⁺ influx and/or release causes mitochondrial Ca²⁺ overload and mitochondrial damage, and ultimately mtDNA release to the cytoplasm causing NLRP3 inflammasome activation.¹⁴¹

Interestingly, by reanalyzing two published datasets^142,143, we found that besides the component of NLRP3 inflammasome (NLRP3 and IL1B), LPS treatment (the well-known priming stimulation) also induces the expression of various ion channels in HUVEC, including Piezo1. A recent study reveals that Piezo1 activation in lung vascular endothelial cells triggers the NLRP3 inflammasome, thereby compromising endothelial barrier function and significantly contributing to psoriasis pathogenesis.¹⁴⁴ Since Piezo1 is upregulated in EC from experimental PH and IPAH patients¹¹, the role of Piezo1 in NLRP3 inflammasome activation in the pathogenesis of PAH will be an interesting research topic.

K⁺ efflux has been recognized as a common necessary event for various NLRP3 activators (e.g., nigericin and α-toxins) to induce NLRP3 inflammasome activation.^145–147 Incubation of primed macrophages in K⁺-free medium is reported to be sufficient to induce NLRP3 inflammasome activation.¹⁴⁷ A decreased cytosolic [K⁺] occurs in cells (e.g., macrophages) treated with NLRP3 activators (e.g., ATP, nigericin, crystalline, particulate matter, cytosolic double-stranded RNA, Staphylococcus aureus hemolysins, and bacterial toxins)^{145,147–150}, whereas blockade of K⁺ efflux by raising extracellular [K⁺] (from 5 to 30–45 mM) significantly inhibit K⁺-efflux associated NLRP3 inflammasome activation.^147,151 Nigericin, an antibiotic derived from Streptomyces hygroscopicus, is a K⁺ ionophore and microbial toxins (e.g., α-toxin from Staphylococcus aureus and Clostridium septicum) and the complement membrane attack complex can form non-selective ion pores/channels in the plasma membrane; all can promote K⁺ efflux based on the electrochemical gradient across the plasma membrane. It is also suggested that the K⁺ efflux through the pores/channels formed by N-GSDMD during the non-canonical inflammasome activation can enhance NLRP3 oligomerization inducing canonical (NLRP3) inflammasome activation (Fig. 5). A rise in [Ca²⁺]_cyt due to receptor-operated and store-operated Ca²⁺ entry as well as IP₃- and Ca²⁺-induced Ca²⁺ release can activate multiple Ca²⁺-activated K⁺ (K_Ca) channels (KCa-Ch) in the plasma membrane and induce K⁺ efflux. In lung vascular cells, there are three classes of K_Ca channels: the large-conductance K_Ca channels (MaxiK or BK channels), intermediate-conductance K_Ca channels (IK) and small-conductance K_Ca channels (SK), with a unitary conductance of 200–300 pS, 30–80 pS, and 5–15 pS, respectively.¹⁵² For example, ATP-mediated activation of P2X channels and P2Y receptors causes Ca²⁺ influx and Na⁺ influx; and the rise in [Ca²⁺]_cyt and membrane depolarization can subsequently induce K⁺ efflux through KCa channels and other types of K⁺ channels (e.g., TWIK2) during ATP-mediated NLRP3 inflammasome activation.¹⁵³ Interestingly, K⁺ efflux through KCNN4 (K_Ca3.1 or IK1), an IK with a unitary conductance of 30–50 pS, participates in Piezo1-enhanced activation of NLRP3 inflammasome.¹⁵⁴

Why and how K⁺ efflux is required for or involved in canonical (NLRP3) inflammasome activation is unclear. In both excitable or non-excitable cells, an enhanced K⁺ efflux results in membrane hyperpolarization close to the K⁺ equilibrium potential of approximately −85 mV and a decrease in intracellular (and nuclear) K⁺ concentration (which is about 140 mM under normal conditions). The high level of cytosolic and nuclear [K⁺] has been demonstrated to be necessary to inhibit intracellular caspase and nuclease activity.^155–157 It is unclear whether cytosolic (and/or nuclear) K⁺ is specifically required to prevent caspase-4/5 and caspase-1 from being activated during inflammasome activation, while the decrease in intracellular [K⁺] due to enhanced K⁺ efflux relieves (or attenuates) the inhibitory effect of K⁺ on caspase-4/5 and caspase-1, thereby causing inflammasome activation. Nevertheless, decreases in K⁺ efflux by raising extracellular K⁺ concentration (i.e., diminishing the driving force for K⁺ efflux) failed to inhibit the activation of caspase-1 and release of IL-1β in macrophages transfected with the constitutively active NLRP3 (NLRP3R258W mutant).¹⁴⁷ These data argue against the direct inhibitory effect of intracellular K⁺ on caspase-1, suggesting that intracellular K⁺, directly or indirectly, targets NLRP3 activation and oligomerization, or the upstream signaling required for activating NLRP3, thereby inhibiting inflammasome activation.

Moreover, membrane hyperpolarization induced by enhanced K⁺ efflux may cause an electrically optimal condition in favor of the induction of NLRP3 inflammasome activation, while membrane depolarization due to blockade of K⁺ efflux (e.g., by high extracellular K⁺ medium) may otherwise create an electric field in favor of the inhibition of NLRP3 inflammasome formation. Recently, a study has indicated that Ca²⁺ influx during NLRP3 activation leads to increased activity of calpain, a Ca²⁺-dependent cysteine protease. Activated calpain releases a pool of caspase-1 sequestered by the cytoskeleton to regulate NLRP3 activation. The “resting” membrane potential is required for the calpain activity, while high K⁺-mediated membrane depolarization or artificial membrane hyperpolarization results in the inhibition of calpain, thus inhibition of NLRP3 and caspase-1 activation. The membrane potential- and Ca²⁺-associated calpain activity may reinforce a mechanism that activates NLRP3 inflammasome independent of intracellular K⁺ or K⁺ efflux.¹⁵⁸ The current knowledge suggests that K⁺ efflux is sufficient, but not necessary, for NLRP3 inflammasome activation.¹⁴¹

Summary and Conclusion: Potential Role in Pulmonary Hypertension

The lungs are an organ that constantly interacts with the external environment through the process of gas exchange. The lung vasculature is under constant mechanical stimulation because of airway radial traction and cardiac output (or RV ejection of blood). Under normal physiological conditions, the lung vascular endothelium primarily exhibits vasodilative and anti-inflammatory effects on the vasculature. Endothelial dysfunction and damage, however, result in pathogenic effects on the lung vasculature, leading to pathological vascular remodeling and pulmonary hypertension. The indirect evidence, obtained from cells and tissues not associated with PH/PAH, that Ca²⁺ signaling engages in controlling cellular senescence and inflammasome activation directs us to speculate that Ca²⁺ influx (and K⁺ efflux) may contribute to the development and progression of PH/PAH. Endothelial Ca²⁺ signaling and intracellular K⁺ homeostasis, facilitated by the orchestrated and coordinated participation of various non-selective and selective cation channels, may potentially play a crucial role in regulating cellular senescence and inflammasome activation in pulmonary arterial endothelial cells and lung microvascular endothelial cells. The senescent EC-initiated SASPs may not only cause secondary senescence in adjacent ECs but also induce cellular phenotypic transitions in adjacent ECs (e.g., endothelial-to-mesenchymal transition) and smooth muscle cells (e.g., contractile-to-proliferative transition) within the vascular wall, leading to vascular remodeling and occlusion. Vascular and perivascular inflammation, because of canonical (NLRP3) and non-canonical inflammasome activation in lung vascular endothelial cells, may potentially be another important pathogenic contributor to the development and progression of vascular remodeling and obliterative intimal lesions in pulmonary hypertension. Targeting endothelial ion channels and membrane receptors involved in the induction of cellular senescence and inflammasome activation is a potentially effective therapeutic strategy for developing new treatments for pulmonary hypertension.

Highlights.

• Endothelial Ca²⁺ signaling and cation channels are involved in inducing cellular senescence and inflammasome activation, both of which are newly identified mechanisms underlying pathological vascular remodeling in pulmonary hypertension (PH).

• Senescent endothelial cells release senescence-associated secretory phenotype (SASP) factors that stimulate the proliferation of surrounding vascular cells and induce vascular remodeling and fibrosis, contributing to the development of PH.

• Endothelial activation of inflammasomes associated with Ca²⁺ influx and K⁺ efflux may be an important mechanism of vascular inflammation and remodeling in PH.

• Differential distribution (or expression level) of mechanosensitive cation channels (MSCC) in pulmonary endothelial and smooth muscle cells suggests a different role of MSCC in different vascular cells in the development and progression of PH.

Non-standard Abbreviations and Acronyms

RV

right ventricle

PVR

pulmonary vascular resistance

PAP

pulmonary arterial pressure

PH

pulmonary hypertension

CO

cardiac output

MSCC

mechanosensitive cation channels

[Ca²⁺]_cyt

cytosolic free Ca²⁺ concentration

SMC

smooth muscle cell

EC

endothelial cell

PAH

pulmonary arterial hypertension

MSC

mesenchymal cells

FB

fibroblasts

myoFB

myofibroblast

SASP

senescence-associated secretory phenotype

ECM

extracellular matrix

COPD

chronic obstructive pulmonary disease

DAG

diacylglycerol

ROCE

receptor-operated Ca²⁺ entry

SOCC

store-operated Ca²⁺ channel

SOCE

store-operated Ca²⁺ entry

VDCC

voltage-dependent Ca²⁺ channel

mtDNA

mitochondrial DNA

PAMP

pathogen-associated molecular pattern

DAMP

damage-associated molecular pattern

PRR

pattern-recognition receptor

LPS

lipopolysaccharide

MAC

complement membrane attack complex

KCa-Ch

Ca²⁺-activated K⁺ (K_Ca) channel

MaxiK or BK

large-conductance K_Ca channel

IK

intermediate-conductance K_Ca channel

SK

small-conductance K_Ca channel