Angiogenesis in lung regeneration and aging

Abstract

Angiogenesis, the formation of new capillary blood vessels, plays a key role in organ development and regeneration, while its dysregulation contributes to disease pathogenesis. In addition to the passive roles of blood vessels in gas exchange and nutrient delivery, endothelial cells (ECs) regulate tissue homeostasis by secreting angiocrine factors that dictate behaviors of surrounding cells to build tissue architecture. In the lung, ECs closely interact with alveolar epithelial cells and other resident cells to coordinate signals necessary for alveolar regeneration. During aging, impaired angiogenic responses diminish tissue regeneration and injury repair, increasing susceptibility to chronic lung diseases. In addition to biochemical signals, dynamic changes in the mechanical forces also regulate pulmonary angiogenesis. In this brief review, we highlight the mechanisms by which ECs maintain vascular homeostasis in the lung and how these processes become dysregulated with aging. We also discuss the impact of changes in the micromechanical environment and summarize recently developed new approaches for investigating EC signaling and cell-cell interactions in the human lung, which leads to the development of future therapeutic strategies.

Graphical Abstract

Introduction

Angiogenesis plays key roles in tissue regeneration^1–8 and repair⁹. Newly formed vasculatures interact with epithelial cells and deliver oxygen, nutrients and cellular components required for tissue regeneration and repair. Tissue-specific endothelium also establishes vascular niches³, secretes angiocrine factors^{3, 8} and promotes regenerative growth following organ resection (e.g., lung^{2, 4–6}, liver¹). Inhibition of EC signaling impairs tissue regeneration and repair^{2, 4–7, 10–13}. In the lung, ECs closely interact with alveolar epithelial cells and other resident cells to coordinate signaling required for alveolar regeneration. Aging is associated with impaired organ function, regeneration, and repair, which increases susceptibility to chronic diseases^14–16. Angiogenic signaling and EC proliferation are attenuated in aging animals^17–22, which is associated with age-related chronic lung diseases such as pulmonary fibrosis and chronic obstructive pulmonary disease (COPD)^{23, 24}. A better understanding of the mechanisms by which aging disrupts angiogenesis in the lung will facilitate the development of more efficient therapies for age-related chronic lung diseases. In this review, we discuss (1) vascular regeneration and repair in the lung, (2) age-associated declines in lung vascular and alveolar regeneration, (3) the role of mechanical forces and extracellular matrix (ECM) in lung angiogenesis, and (4) recently developed state-of-the-art methods for investigating human lung EC signaling and physiology.

I. Vascular regeneration and repair in the lung

Pulmonary vasculature

The pulmonary circulation is a low-pressure and high-flow system, which is tightly regulated by the lung vasculature²⁵. Pulmonary blood vessels are subdivided into proximal (pulmonary arteries (PAs) and veins) and peripheral regions (arterioles and capillaries), each characterized by distinct microenvironments. For example, luminal pressure decreases from ~15–20 mmHg in the main PA to ~5 mmHg in distal arterioles and capillaries, generating a steep vascular pressure gradient. Proximal vessels are enriched in elastin with thick smooth muscle layers, conferring high compliance, whereas distal arterioles contain less elastin and more collagen. Pulmonary capillaries, which line the alveolar surfaces, lack smooth muscle layers and consist primarily of a thin basal lamina with pericyte-associated ECM, including collagens, laminins, nidogens, and heparan sulfate proteoglycans²⁶, facilitating efficient gas exchange. Under physiological conditions, wall shear stress in the pulmonary arterial tree ranges 15–20 dynes/cm², but is significantly reduced in the pathological conditions such as pulmonary hypertension (PH)²⁷. These architectural and mechanical features are transduced into biological signals that are necessary for maintaining lung vascular structure and function.

Angiogenesis in lung regeneration and repair

In addition to serving as structural components of the semi-permeable capillary wall, pulmonary capillary ECs establish vascular niche and secrete angiocrine factors to maintain lung alveolar homeostasis^{2–6, 10–13}. EC-derived angiocrine factors also determine alveolar stem cell fate and immune responses to coordinate lung regeneration and repair^{3, 4, 8, 28}. For example, induction of matrix metalloproteinase 14 (MMP14) expression in pulmonary capillary ECs following pneumonectomy (PNX) stimulates epithelial progenitor cell proliferation and alveolarization, promoting alveolar regeneration⁴. WNT signaling is also one of the major driving factors for angiogenesis^{29, 30}; knockdown of the WNT co-receptor low-density lipoprotein receptor-related protein 5 (Lrp5) in ECs inhibits post-PNX regenerative lung growth by suppressing expression of the angiogenic receptor TIE2 expression⁶. Similarly, EC-specific knockdown of the Hippo pathway effector yes-associated protein 1 (Yap1), which controls angiogenesis in multiple organs (e.g., lung, bone, eye^{2, 21, 31–33}), impairs EC regeneration by decreasing TIE2 expression in the mouse PNX model². Although angiogenesis is necessary for lung regeneration and repair, the effects of angiogenic signaling are context dependent. For example, depletion of vascular endothelial growth factor receptor 1 (VEGFR1) in ECs enhances differentiation of type II alveolar epithelial cells (AT2 cells), promoting repair in pulmonary fibrosis through secretion of soluble factors and ECM components¹⁰. Conversely, thrombospondin 1 (TSP1), an inhibitor of angiogenesis³⁴, facilitates bronchioalveolar stem cell differentiation during regenerative lung growth and injury repair⁷. The responses to angiocrine factors vary across cell types and contexts during lung regeneration and repair.

While EC-derived angiocrine factors contribute to lung regeneration and repair^{4, 8, 28}, disruption of angiocrine signaling promotes lung pathology^{2, 6, 35–38}. For example, EC–specific deletion of methyltransferase-like 3 (Mettl3) and forkhead box protein O1 (Foxo1) in the lung impairs post-PNX lung growth and exacerbates fibrosis by promoting intussusceptive angiogenesis in mice³⁹. In addition, suppression of dopaminylation of the metabolic enzyme triosephosphate isomerase 1 (TPI1) in ECs induces ferroptosis-associated angiocrine signaling that aberrantly activates fibroblasts, shifting lung repair toward fibrosis⁴⁰.

EC heterogeneity in lung regeneration and injury repair

In adult lungs, capillary type 1 ECs (CAP1s) exhibit stem-like properties and re-enter the cell cycle following injury. In an elastase-induced mouse lung injury model, CAP1s exhibit progenitor-like state characterized by expression of the apelin receptor (APLNR) and protein C receptor (PROCR) at the early stage after injury, differentiating into capillary type 2 ECs (CAP2s) at the later stage of repair⁴¹. CAP1s also contribute to repair from lipopolysaccharide (LPS)-induced lung injury. At early time points after LPS challenge (within 6 hours), CAP1s transition into distinct regenerative subpopulations marked by co-expression of apelin (APLN) and PROCR, genes associated with endothelial activation and progenitor-like function^{13, 42}, which is followed by the emergence of a proliferative Mki67/forkhead box protein M1 (Foxm1)-positive subpopulation at the later stage (day 3 after LPS challenge)¹³. In addition, a subset of CAP1s expressing activating transcription factor 3 (ATF3), a stress-responsive regulator of metabolism and immunity, proliferates following influenza-induced injury and restores alveolar structure¹². Loss of endothelial Atf3 impairs vascular regeneration by increasing EC apoptosis, which reduces epithelial and CAP2 density and leads to persistent emphysema-like alveolar enlargement¹². Thus, CAP1s represent a key EC subtype that drives vascular regeneration and injury repair across multiple lung injury models through distinct signaling pathways. In addition to CAP1s and CAP2s, recent studies suggest that venous ECs also contribute to lung injury repair⁴³; venous ECs exhibit plasticity and can transdifferentiate into CAP1s and CAP2s to replenish the vascular plexus. Coordinated spatiotemporal regulation of angiogenic signaling in multiple EC subtypes may be necessary for effective lung regeneration and repair.

Lung EC subtypes also contribute to injury repair in the neonatal stage. Depletion of CAP1s compromises vascular repair capacity in a hyperoxia-treated mouse model of bronchopulmonary dysplasia (BPD), leading to subsequent pulmonary vascular diseases. In contrast, CAP2s are increased in hyperoxia-treated mouse lungs and exhibit a pathological gene expression profile, including upregulation of pro-inflammatory genes (e.g., connective tissue growth factor (Ctgf), FXYD domain containing ion transport regulator 5 (Fxyd5)) and anti-angiogenic genes (e.g., cyclin dependent kinase inhibitor 1A (Cdkn1a), tissue inhibitor of metalloproteinases 3 (Timp3), serpin family E member 1 (Serpine1), insulin like growth factor binding protein 7 (Igfbp7))⁴⁴. However, CAP2s are reduced in the nitrofen-exposed rat model of congenital diaphragmatic hernia, another neonatal lung developmental disorder⁴⁵. Disruption of the balance between CAP1s and CAP2s, along with aberrant gene expression, impairs injury repair and contributes to lung developmental disorder. Recent single-cell RNA sequencing (scRNA-seq) analyses have identified neurotrophic receptor tyrosine kinase 2 (NTRK2), a regulator of EC proliferation and migration, as a key factor in neonatal lung developmental disorder and injury repair^46–48. In a hyperoxia-treated mouse model of BPD, Ntrk2 expression is suppressed in CAP1s, while Apln, normally restricted to CAP2s, becomes aberrantly expressed in CAP1s⁴⁶. Furthermore, the full-length isoform of NTRK2 (NTRK2-FL) promotes CAP1 repair in a hyperoxia-induced mouse BPD model, whereas the truncated isoform (NTRK2-T1) drives maladaptive responses and persistent alveolar simplification, suggesting that imbalance between NTRK2 isoforms (NTRK2-FL vs. NTRK2-T1) contributes to endothelial dysfunction after hyperoxic injury⁴⁷. In addition to neonatal hyperoxic injury, NTRK2-T1 is upregulated in CAP1s in multiple adult lung injury models, including LPS treatment, sendai virus and H3N2 influenza infection, suggesting that NTRK2 pathway is involved in adult lung pathologies as well⁴⁸.

In addition to ECs, immune cells also play important roles in lung alveolar regeneration. Local and bone marrow-derived myeloid cells (e.g., monocytes, macrophages) stimulate proliferation of AT2 progenitor cells to promote post-PNX lung growth⁴⁹. Disruption of immune cell signaling contributes to pulmonary fibrosis. Monocyte-derived alveolar macrophages, but not tissue-resident alveolar macrophages, drive fibrotic remodeling during the acute phase of bleomycin-induced lung injury^{50, 51}. Neutrophils also contribute to acute lung inflammation and fibrosis by releasing pro-inflammatory mediators, including elastase⁵², MMPs⁵³ and neutrophil extracellular traps⁵⁴. ScRNA-seq analyses have indicated that alveolar macrophages are a heterogeneous population with distinct functions in maintaining lung parenchyma^{50, 51}. Thus, selectively targeting alveolar macrophage differentiation, rather than broadly depleting monocyte-derived or tissue-resident alveolar macrophages, may represent a more effective strategy to ameliorate fibrosis^{50, 51}. Importantly, ECs modulate immune cell activation, polarization, and survival through the paracrine signaling mechanisms^{55, 56}. For example, CAP1s secrete transforming growth factor beta 1 (TGF-β1), promoting differentiation of monocytes into interstitial macrophages⁵⁷. In response to LPS, ECs secrete the WNT activator R-spondin3, which promotes transition of interstitial macrophages toward an anti-inflammatory phenotype to facilitate resolution of inflammation⁵⁸. Pulmonary ECs and specific EC subpopulations orchestrate immune responses to promote tissue regeneration and repair.

II. Age-associated decline in lung vascular and alveolar regeneration

Recent advances in aging research have identified twelve hallmarks of aging, including epigenetic alterations, mitochondrial dysfunction, telomere attrition, cellular senescence, chronic inflammation, genomic instability, loss of proteostasis, impaired autophagy, deregulated nutrient sensing, stem cell exhaustion, altered intercellular communication, and dysbiosis⁵⁹. Among them, this review focuses on recent findings demonstrating how epigenetic alterations, oxidative stress and mitochondrial dysfunction, telomere attrition, cellular senescence, and chronic inflammation contribute to age-dependent changes in lung vascular and alveolar regeneration (Fig. 1). We also highlight age-related alterations in EC signaling in the lung and the role of EC dysfunction in age-associated lung diseases.

Fig. 1. Pulmonary EC dysregulation during aging.

Proper angiogenic signaling is essential for lung vascular and alveolar regeneration. Age-related dysregulation of key biological processes in ECs, including epigenetic alterations, oxidative stress and mitochondrial dysfunction, telomere attrition, cellular senescence, and chronic inflammation leads to a progressive decline in angiogenic capacity in the aged lung. Age-dependent changes in the ECM components and disruption of ECM structures, which alter cellular responses to mechanical forces, also impair angiogenic signaling. In addition to alveolar epithelial cell dysfunction and excessive fibroblast proliferation, EC dysregulation is one of the major contributors to the pathogenesis of age-related lung diseases, including COPD and pulmonary fibrosis.

Epigenetic alterations

Epigenetic alterations modify chromatin accessibility and gene expression programs, leading to impaired angiogenic signaling and EC dysfunction⁶⁰. Epigenetic alterations accumulate during aging and contribute to cellular dysfunction and the pathogenesis of age-related diseases. For example, suppression of FOXM1 due to promoter hypermethylation impairs lung EC regeneration and increases mortality following sepsis in aged mice⁶¹. In contrast, pharmacologic or genetic restoration of FOXM1, or treatment with DNA demethylating agents that reactivate FOXM1, restores EC regeneration, normalizes vascular repair and resolution of inflammation, and improves survival in aged mice after sepsis challenge⁶¹. Similarly, the transcription factor ETS-related gene (ERG), which is critical for maintaining EC identity and promoting vascular repair, is epigenetically repressed in aged ECs, contributing to fibroblast activation and impaired resolution of pulmonary fibrosis⁶².

Oxidative stress and mitochondrial dysfunction

Oxidative stress and mitochondrial dysfunction accumulate with age and impair EC metabolism, compromising angiogenic responses. Age-associated mitochondrial insufficiency driven by impaired electron transport, reduced mitophagy, and accumulation of mitochondrial DNA damage leads to a self-amplifying cycle, in which oxidative stress further disrupts mitochondrial function, promoting chronic inflammation, cellular senescence, and age-related tissue dysfunction. Although the direct contribution of the pulmonary endothelial reactive oxygen species (ROS)–mitochondrial axis to lung aging has not been fully defined, the ROS–mitochondrial axis represents a key hallmark of aging⁶³ and is likely involved in the age-associated decline in regenerative ability in the lung. For example, elevated ROS levels, together with disturbed antioxidant defenses, drive the development of age-related lung diseases such as pulmonary fibrosis, pulmonary arterial hypertension (PAH) and COPD⁶⁴. In contrast, activation of the SUMO-specific protease 1 (SENP1)-Sirtuin 3 (SIRT3) axis attenuates mitochondrial redox signaling, promotes AT2 cell proliferation and their differentiation into AT1 cells, and suppresses fibrotic responses⁶⁵.

Aging impairs the activation of key metabolic regulators, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and AMP-activated protein kinase (AMPK), while activating mechanistic target of rapamycin kinase C1 (mTORC1), all of which are essential for maintaining energy homeostasis and vascular remodeling. For example, age-associated upregulation of TWIST1 impairs regenerative lung growth by inhibiting endothelial PGC1α¹⁷. Pulmonary EC–specific deletion of Ppargc1a, encoding PGC1α, suppresses angiogenesis in a hypoxic lung and exacerbates PH, accompanied by increased oxidative stress, cellular senescence, and DNA damage⁶⁶. Importantly, PGC1α is regulated by AMPK, an energy-sensing kinase that coordinates cellular metabolism, mitochondrial function, and stress responses to maintain tissue homeostasis⁶⁷. Inactivation of endothelial AMPK accelerates lung fibrosis, whereas pharmacologic activation of AMPK by metformin attenuates fibrosis⁶⁸. Mechanistically, loss of AMPK activates YAP signaling and induces the angiocrine factor plasminogen activator inhibitor type 1 (PAI-1), which stimulates fibrosis in human lungs⁶⁸. In contrast, age-associated activation of mTORC1 and its downstream effector S6K1 induces nitric oxide synthase 3 (eNOS) uncoupling, leading to endothelial dysfunction to promote vascular aging⁶⁹. Although pulmonary endothelial mTOR signaling is required to maintain microvascular niche homeostasis, excessive activation of mTOR in lung ECs decreases angiogenic potential, leading to emphysema and accelerated lung aging⁷⁰. Thus, metabolic signaling pathways in ECs are kay mediators of lung vascular aging. It remains incompletely understood how age-related metabolic disruption in ECs contributes to lung injury repair and the progression of chronic lung diseases. Recent single-cell transcriptomic analyses of murine choroidal and lung tumor ECs reveal endothelial metabolic heterogeneity and plasticity during angiogenesis, identifying conserved metabolic genes, including squalene epoxidase (Sqle) and aldehyde dehydrogenase 18 family member A1 (Aldh18a1), that are upregulated across diseases and tissues⁷¹. Further investigation into age-dependent changes in endothelial metabolic heterogeneity is warranted.

Telomere attrition

Telomere length progressively shortens with each mitotic division, and telomere attrition is one of the core hallmarks of cellular aging associated with chronic diseases. Telomere dysfunction has been linked to impairment of alveolar stem cell function⁷². In mouse models, telomere shortening induced by knockout of telomerase components increases the formation of telomere-associated DNA damage response foci, triggers inflammation, and recapitulates key features of idiopathic pulmonary fibrosis (IPF)⁷³. Consistently, AT2 cell-specific depletion of the telomere-binding proteins telomeric repeat-binding factor 1 (TRF1) or TRF2 impairs alveolar regeneration and promotes inflammation and fibrosis^{72, 74}. Telomere-associated DNA damage foci are also increased in small airway epithelial cells from patients with COPD and in aged mouse lung, and the effect is exacerbated by cigarette smoke exposure through increased ROS production and secretion of inflammatory cytokines⁷⁵. Similarly, telomerase RNA component (Terc) null mice, which harbor dysfunctional telomeres, exhibit early disruption of alveolar structure, suggesting the role of telomere dysfunction in lung aging and COPD pathogenesis⁷⁵.

Regarding the relationship between angiogenesis and telomere attrition, it has been demonstrated that inflammation and oxidative stress, conditions associated with aberrant angiogenesis, accelerate telomere shortening⁷⁶. Although no direct correlation has been established between telomere length and levels of the major angiogenic factor vascular endothelial growth factor A (VEGFA), recent study suggests that telomere attrition during aging may be driven by increased hematopoiesis and platelet production⁷⁷.

EC senescence

Cellular senescence is a state of irreversible cell cycle arrest characterized by key molecular changes, including p53, p21, and p16^78–80. Senescent cells exhibit a distinct secretory profile known as the senescence-associated secretory phenotype (SASP), characterized by the release of pro-inflammatory cytokines, chemokines, growth factors, proteases, MMPs, and ECMs^{81, 82}. Cellular senescence is induced by various stressors such as DNA damage, telomere shortening, oxidative stress, and oncogenic activation^78–80. For example, persistent DNA damage at telomeres triggers a sustained DNA damage response (DDR) that promotes cellular senescence and SASP-mediated inflammation during aging^83–85. Telomerase reverse transcriptase (TERT) reduces the expression of senescent and inflammatory markers, restores nitric oxide (NO) production, and promotes angiogenesis⁸⁶. Similarly, TERT reduces senescent cell accumulation in the lung, preserves pulmonary capillary density by promoting proliferation of CD34⁺ endothelial progenitor cells in aged mice, and prevents emphysema development in a Sugen-hypoxia model (VEGFR inhibition under chronic hypoxia)⁸⁷. In addition, DDR regulators ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) activate GATA4, which in turn stimulates nuclear factor kappa B (NF-kappaB) signaling to initiate the SASP and promote senescence, contributing to aging and age-associated inflammation⁸⁸.

Senescent cells accumulate with aging, impair stem cell differentiation, and inhibit organ regeneration and repair, contributing to age-related functional decline and increased mortality^78–80. Cellular senescence occurs across diverse cell types in the aged lung. However, while only about 10% of p16⁺ senescent cells express the epithelial marker mucin 1 (MUC1), approximately 30% express the endothelial marker CD31, indicating preferential accumulation of senescent ECs in the aged lung⁸⁷. Senescent ECs exhibit characteristic morphological changes, including altered cell shape and size, compromised nuclear integrity, and resistance to apoptosis^{21, 89–91}, and contribute to pathological processes associated with progressive EC dysfunction^{19, 21}.

Regarding signaling mechanisms, NADPH oxidase (NOX) has been reported to drive DNA damage and promote inflammation, tissue dysfunction, and cellular senescence; in contrast, inhibition of NOX1 rescues age-dependent impairments in blood flow, angiogenesis, vasodilation, and EC wound responses by disrupting a NOX1- interleukin-6 (IL-6) pro-inflammatory signaling loop⁹². In addition, nitrilase family member 2 (NIT2), a redox-sensitive enzyme that converts α-ketoglutaramate (αKGM) to α-ketoglutarate (αKG), senses oxidative stress to control glutamine metabolism. EC-specific knockout of Nit2 in mice increases intracellular αKGM levels, induces cellular senescence, and impairs angiogenesis⁹³. In the lung, age-associated increases in EC senescence disrupt cell-cell junctional integrity through downregulation of VE-cadherin and zona occludens 1 (ZO-1), impairing barrier function to promoting immune cell trafficking and inflammation⁹⁴.

Although senescent cells are often considered detrimental, they also play essential roles in tissue repair and wound healing⁹⁵. For example, p16⁺ senescent fibroblasts and ECs emerge at early stages following cutaneous wound and are required for efficient wound closure through secretion of platelet-derived growth factor AA (PDGF-AA)⁹⁵. The cyclin-dependent kinase inhibitors p21 and p16 have long been recognized to drive shared senescence-related functions; however, recent studies have revealed substantial heterogeneity between p21- and p16- expressing senescent cells across different cell types and tissues, including distinct SASP profiles and trajectory dynamics^{96, 97}. Further investigation of EC senescence heterogeneity in lung angiogenesis will provide mechanistic insight into age-related disruption of angiogenesis.

Chronic inflammation

Immune cells and inflammatory reaction are essential for protecting the body against acute infections, as well as for tissue/organ regeneration and injury repair. Immune system dysfunction is one of the hallmarks of aging. Age-dependent alterations in pulmonary EC signaling exacerbates inflammatory lung injury, which disrupts endothelial barrier integrity and skews EC function toward a pro-inflammatory and pathological angiogenic phenotype, leading to pulmonary edema, sustained inflammation and fibrosis in aged lung⁹⁸. For example, endothelial FOXM1 promotes vascular repair and resolution of inflammation following sepsis-induced lung injury, while age-associated decline in FOXM1 expression impairs the effects and results in increased mortality in aged animals⁶¹. Age-dependent changes in innate and adaptive immunity involve two opposing yet complementary interconnective processes: immunosenescence, characterized by a decline in protective immune functions, and inflammaging, defined as chronic, low-grade sterile inflammation⁹⁹.

Immunosenescence is a progressive decline in the function of immune organs (e.g., thymus, spleen, and skin), characterized by impaired physical and mucosal barriers, reduced hematopoietic stem cell function, and weakened innate and adaptive immune responses. EC signaling is one of the key contributors for immunosenescence. scRNA-seq analyses of splenic ECs from young and old mice reveal age-dependent changes in endothelial heterogeneity, reprogramming, and gene expression patterns associated with immunosenescence¹⁰⁰. Studies using humanized K18-hACE2 young vs. aged mouse models have also demonstrated that aged lung ECs exhibit attenuated inflammatory signaling (e.g., interferon and Toll-like receptor) and suppressed cytokine/chemokine responses that normally protect against acute inflammation, which contributes to increased mortality in COVID-19 pneumonia in aged mice¹⁰¹. However, immune cell senescence has time- and context-dependent beneficial roles¹⁰². During the early phase of hyperoxia-induced lung injury in neonatal mice, transition of macrophage subpopulations expressing senescence markers toward M2-like phenotype facilitates tissue remodeling and resolution of inflammation¹⁰². These senescent M2 macrophages are subsequently engulfed by healthy macrophages and cleared from the lungs¹⁰². Thus, the timing and cell-type specificity of immune cell senescence critically regulate postnatal lung development and injury repair.

Inflammaging is a chronic, low-grade sterile inflammatory state driven by the accumulation of senescent cells, SASP factor secretion, metabolic dysregulation, mitochondrial dysfunction, and gut dysbiosis. These processes elevate circulating cytokines, including IL-6, tumor necrotic factor-alpha (TNF-α), and interferon-gamma (IFN-γ), exacerbating tissue damage and cellular dysfunction⁹⁹. A murine model of EC senescence reveals that excessive EC production of C-X-C motif chemokine ligand 1 (CXCL1) promotes pathogenic neutrophil adhesion to senescent endothelium in inflamed skin and lung¹⁰³. Increased expression of senescent markers in aged mouse lung ECs is also associated with enhanced inflammatory responses, including upregulation of intercellular adhesion molecule 1 (ICAM1), which destabilizes cell-cell junctions, impairs alveolar barrier function, and promotes neutrophil extravasation and chronic inflammation in the aged lung⁹⁴.

These two complementary immune dysfunctions – immunosenescence and inflammaging- synergistically increase susceptibility to infection and contribute to the progression of age-related cardiovascular and pulmonary disorders¹⁰⁴. Further investigation of age-associated alterations in EC signaling underlying immune dysfunctions may lead to more efficient strategies for chronic lung diseases in the elderly.

Angiogenic signaling and ability during EC aging

Age-related biological and chemical dysregulation leads to a progressive decline in angiogenic capacity in the aged lung, contributing to age-related lung diseases, including pulmonary fibrosis and COPD, as well as impaired recovery from acute lung injury^{94, 101, 105, 106}. Structurally, aged lungs are characterized by enlarged airspaces, reduced surface area, and diminished elastic recoil, accompanied by disorganized alveolar capillaries and reduced EC number and angiogenic ability¹⁹.

During aging, angiogenic signaling becomes also disrupted and capillary ECs progressively lose their ability of vascular formation. For example, vascular formation is significantly declined in the Hutchinson-Gilford progeria syndrome, a premature aging disorder caused by the lamin A/C (LMNA) mutation by downregulating serum response factor (SRF)¹⁰⁷. A blood vessel organoid model with LMNA-mutant human embryonic stem cells reveals the vascular defects and suppression of expression of SRF and angiogenesis-related genes, while overexpression of SRF rescues endothelial function in the organoids¹⁰⁷. In addition, a transcription factor, TWIST1, which contributes to the pathogenesis of age- and angiogenesis-related diseases such as pulmonary fibrosis and atherosclerosis, is higher in ECs isolated from aged human adipose tissues and mouse lungs compared to those from young tissues and mediates age-dependent decline in angiogenesis¹⁷. Knockdown of TWIST1 in aged human ECs increases the levels of PGC1α and VEGFR2, and restores EC proliferation and migration, while inhibition of PGC1α suppresses these effects¹⁷, suggesting that upregulation of endothelial TWIST1 mediates age-dependent decline in angiogenesis.

EC and alveolar epithelial interaction in lung regeneration/repair during aging

With aging, endothelial–epithelial crosstalk becomes dysregulated due to EC dysfunction and cellular senescence, leading to impaired epithelial regeneration and pro-inflammatory response. Post-PNX regenerative lung growth, which is partly driven by EC signaling, is impaired in aged mouse lung^{17, 19}. Inhibition of WNT signaling reduces EC survival and impairs angiocrine support for epithelial repair in the aged lung¹⁰⁸. Endothelial aging suppresses WNT-LRP5 signaling, resulting in reduced angiopoietin1 (ANGPT1)-TIE2 signaling, disrupted angiogenesis, and impaired post-PNX lung vascular and alveolar regeneration¹⁹. Knockdown of Lrp5 in ECs further exacerbates impairment of angiogenesis and alveolar morphogenesis in the aged lung, suggesting that EC LRP5 is required for maintaining vascular and alveolar morphogenesis in aging lungs¹⁹.

In contrast, anti-angiogenic TSPs, particularly TSP1³⁴, accumulate with age and inhibit angiogenesis by inducing EC senescence via CD47 and NOX1 signaling^{109, 110}. Although TSP1 promotes alveolar epithelial cell regeneration following lung injury⁷, excessive TSP1 may lead to activation of TGF-β, which can be detrimental. Indeed, levels of TSP1 and TGF-β are elevated in the lungs of preterm infants at risk for BPD¹¹¹. Thus, increased levels of TSP1 in the aged lung may impair angiogenesis and reduce repair ability. In addition, circulating levels of another anti-angiogenic factor endostatin increase with aging¹¹² and are negatively associated with outcomes in PAH¹¹³ and COVID-19 pneumonia¹¹⁴.

Age-dependent disruption of transcriptional programs in ECs (e.g., YAP/TAZ, TWIST) also contributes to impaired vascular and alveolar regeneration^{17, 21, 115}. TWIST1 expression is increased in aged lung ECs and age-dependent inhibition of post-PNX lung growth is suppressed in Tie2-specific Twist1 conditional knockout mouse lungs, in which VEGFR2 expression in ECs increases after PNX¹⁷. Recent scRNA-seq and spatial transcriptomics analyses of bleomycin-treated mouse lungs across time points demonstrate that aging impairs the reprogramming of pulmonary CAP1s and CAP2s after bleomycin challenge, which exhibits temporally and spatially mismatched endothelial and epithelial regeneration, leading to pro-fibrotic and pro-inflammatory features and incomplete tissue resolution¹⁰⁵. Age-related alterations in pro- and anti-angiogenic signaling and endothelial–epithelial interaction along with changes in specific EC subpopulations, interfere with lung progenitor differentiation and disrupt vascular function, which compromises alveolar homeostasis, lung function, as well as regenerative and repair ability in the lung, contributing to the progression of chronic lung diseases. In fact, human lung function is suppressed approximately 1% per year after the age of 35¹¹⁶.

In this section, we highlight several major hallmarks of aging and age-dependent changes in angiogenic signaling. Although other hallmarks of aging⁵⁹ may also contribute to age-dependent decline in vascular and alveolar regenerative capacity and lung diseases, their specific roles and underlying mechanism in lung vascular regeneration have not been fully investigated. Further studies are needed to elucidate these mechanisms.

EC dysfunction in age related lung diseases

Age-associated pulmonary EC dysfunction and maladaptive angiogenic responses promote chronic inflammation, vascular remodeling, and fibrotic progression, contributing to age-associated pulmonary pathologies such as PH, COPD, and IPF.

PH is frequently associated with age-related chronic lung diseases, including IPF and COPD. In PH, EC senescence is closely linked to pathological vascular remodeling, characterized by uncontrolled proliferation and accumulation of PA smooth muscle cells (PASMCs) to distal PAs^{81, 117, 118}. ECs isolated from patients with PAH (WHO group1 PH, which includes idiopathic and heritable PH) exhibit increased senescence-associated β-galactosidase (SA-β-gal) activity and elevated expression of senescence markers such as p21 and p16 compared to healthy controls^{81, 117}. Hypoxia-induced PASMC accumulation to PAs is attenuated in EC-specific p16 knockout mice, in which TWIST1-PDGFB signaling that promotes PASMC proliferation and accumulation to distal PAs^{81, 119} is suppressed⁸¹. Furthermore, EC senescence impairs the reversibility of PH in rat PH models¹¹⁸, suggesting a role of EC senescence in the irreversible progression of end-stage PAH. However, the role of senescence appears to be context dependent. Elimination of senescent cells in p16-ATTAC mice or through senolytic strategies have been reported to worsen pulmonary hemodynamics and exacerbate PASMC accumulation to distal PAs¹²⁰. The effects of cellular senescence may vary across cell types or distinct senescence-associated pathways (e.g., p21, p53) may differentially contribute to PH pathology. Accumulating evidence suggests that PH severity correlates with aging¹²¹. PAH patients exhibit reduced responses to therapy and worsen outcomes during aging, while the prevalence of WHO group 2 PH (PH due to left heart disease) and group 3 PH (PH due to lung diseases and/or hypoxia) increases in the aging population¹²¹. Further studies leveraging scRNA-seq to define senescence gene profiles in different lung cell types and age groups will elucidate the mechanism.

COPD is another age-associated disorder characterized by persistent and progressive airflow obstruction. Although destruction of alveolar walls and alveolar epithelial cell dysfunction are major characteristics of COPD, EC dysfunction, EC apoptosis and capillary rarefaction also play critical roles in impaired gas exchange and contribute to the progressive decline in pulmonary function in COPD patients^{23, 122}. EC senescence driven by telomere shortening and dysfunction has been shown to promote lung inflammation, thereby exacerbating COPD severity in the aged lung¹²³. Vascular rarefaction and alveolar regression can be rescued through intravenous delivery of healthy lung ECs¹²⁴ or by suppression of an emphysema driver gene leucine-rich α−2-glycoprotein-1 (LRG1), which mediates neovascularization through TGF-β1/activin receptor-like kinase 1 (ALK1) signaling in ECs¹²⁵, highlighting the therapeutic potential of EC-based cell and gene therapies to restore vascular function in COPD.

In addition to alveolar epithelial disfunction and excessive fibroblast proliferation, aberrant angiogenesis is also associated with IPF, where structurally and functionally aberrant vasculature is concentrated within and surrounding fibrotic foci, disrupting alveolar gas exchange^{126, 127}. Dysfunctional ECs fail to properly coordinate epithelial remodeling, leading to defective tissue repair in the fibrotic lung^{106, 128}. Aging lungs exhibit sustained endothelial activation in response to bleomycin challenge by activating YAP/TAZ signaling in atypical chemokine receptor 1⁺ (Ackr1⁺) venous and tyrosine receptor kinase B⁺ (Trkb⁺) capillary ECs. This dysregulation disrupts capillary morphogenesis and promotes fibrosis, indicating that aging-related EC dysfunction suppresses lung injury repair and leads to persistent fibrotic remodeling¹⁰⁶. Recent studies using bleomycin-induced pulmonary fibrosis models also demonstrate that aged mice exhibit capillary rarefaction, increased expression of pro-fibrotic genes, and reduced expression of vascular homeostasis gene (e.g., eNOS), whereas young mice show increased capillary density following bleomycin challenge¹²⁹. Transcriptional analyses of ECs from bleomycin-treated young or aged fibrotic mouse lungs further reveal that age-dependent decreases in ERG expression and function impair vascular repair, resulting in persistent fibrosis⁶². In addition, sustained TWIST1 activation in ECs, as observed in aged lung¹⁷, contributes to bleomycin-induced pulmonary fibrosis by stimulating ANGPT-Tie2 signaling¹³⁰. Further understanding of how pulmonary ECs drive disease pathobiology in the context of aging may lead to improved therapeutic strategies for chronic lung diseases.

III. Mechanical forces and ECM in lung angiogenesis

The pulmonary microenvironment is mechanically dynamic. ECM composition and mechanical properties, as well as multiple mechanical forces, including shear stress, cyclic stretch, and hydrostatic pressure govern EC and other resident cell behaviors in the lung. ECs, located at the interface between vasculature and alveolar epithelial cells, sense and respond to these physical forces in a highly specialized manner.

ECM in lung angiogenesis and aging

ECM and mechanical stimuli play critical roles in guiding vascular patterning, EC signaling, and angiogenesis during development and regeneration^{131, 132}. ECM composition and mechanical properties are altered during aging¹³³, impairing endothelial function and reducing angiogenic ability^{19, 21}. Major lung ECM proteins, including collagen IV, laminin, fibronectin, and elastin form an organized basement membrane and provide structural support as well as biochemical and mechanical signals that control EC migration, proliferation, and sprouting morphogenesis, thereby guiding alveolar capillary formation and stability. ECs sense ECM composition and stiffness through integrin-mediated interactions and transduce mechanical signals via intracellular pathways (e.g., focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K)-AKT, mitogen-activated protein kinase (MAPK)) to regulate EC behaviors. Changes in ECM composition and mechanical properties further modulate how ECs respond to mechanical forces, coordinating EC responses in a spatiotemporal manner¹³⁴. For example, collagen VI, an ECM protein that impacts cell mechanical responses¹³⁵, is enriched at the peripheral region of post-PNX mouse lungs, where ECs differentially sense mechanical forces and upregulate focal adhesion protein paxillin expression, promoting EC regeneration¹³⁴. In addition, ECM serves as a reservoir for angiogenic growth factors (e.g., ANGPTs, VEGF, fibroblast growth factor (FGF)), modulating their spatial and temporal availability during vascular remodeling.

As the lung ages, the ECM undergoes profound compositional and structural remodeling, altering mechanical properties of the lung and intracellular signaling¹³³. There is increased deposition of fibrillar collagens (e.g., type I and III), accompanied by a reduction and disorganization of basement membrane components such as collagen IV and laminins, disrupting epithelial–endothelial interactions^{136, 137}. Elastic fiber networks also become fragmented due to reduced elastin synthesis, increased mechanical stress, and elevated protease activity, leading to impaired elastic recoil¹³⁸. In addition to these compositional changes, aging enhances ECM crosslinking dynamics through lysyl oxidase (LOX) and LOX-like enzymes (LOXL1–4), which catalyze the crosslinking of collagen and elastin. LOX-mediated modulation of ECM stiffness has been demonstrated to restore EC junctional integrity in LPS-treated mouse lungs and support lung vascular development in a hyperoxia-treated neonatal mouse BPD model^{132, 139}. In a rat PH model, elastin degradation, coupled with the accumulation of fibrillar collagens, contributes to pathological PA remodeling, while preservation of elastic fibers reduces inflammation and collagen accumulation and normalizes right ventricular and pulmonary hemodynamics¹⁴⁰. Although LOX/LOXL activity is increased and ECM stiffness is elevated in aging and fibrotic lungs, other studies report diminished LOX function during aging^{141, 142}, suggesting that LOX activity is context dependent and may vary in different diseases and during aging.

The aged ECM also accumulates advanced glycation end products (AGEs), which further disrupts protein structure and ligand-receptor interactions¹⁴³. Age-dependent increases in oxidative stress and chronic inflammation promote non-enzymatic glycation of ECMs, leading to AGE accumulation in the lung¹⁴⁴. These AGEs bind to their receptors (RAGE) to activate intracellular signaling, while also irreversibly crosslinking ECM proteins, increasing tissue stiffness, and reducing matrix degradability, thereby altering ECM structure in the aged lung¹⁴⁵. These changes feedback to further promote oxidative stress and inflammatory signaling in ECs, disrupting matrix-integrin signaling and reducing angiogenesis and regenerative ability¹⁴⁶. These age-associated ECM component and structural changes -stiffer and denser ECMs- compromise the micromechanical environment in the lung, which inhibits vascular regeneration and promotes maladaptive remodeling in the aged lung, ultimately contributing to age-associated chronic lung diseases.

Effects of mechanical force on lung angiogenesis during aging

Lung cells are continuously exposed to different types of mechanical forces. Alveolar epithelial cells undergo cyclic stretch during breathing, whereas vascular ECs are exposed to shear stress from blood flow, cyclic strain, and transmural pressure. Mechanical stretch promotes post-PNX alveolar regeneration; increased mechanical tension following unilateral PNX stimulates CDC42-dependent actin remodeling, which activates c-Jun N-terminal kinase (JNK), p38, and YAP in AT2 cells, thereby driving alveolar regeneration¹⁴⁷. Mechanical tension stimulates EC proliferation more in the peripheral region¹³⁴, where lung structural deformation is more pronounced after PNX¹⁴⁸. In addition, pulmonary arterial pressure increases following PNX, activating YAP1 in ECs and promoting angiogenesis through upregulating TIE2 expression¹⁴⁹.

ECs sense mechanical forces through specialized mechanosensors, including platelet and endothelial cell adhesion molecule 1 (PECAM1), VE-cadherin, integrins, and mechanosensitive ion channels such as PIEZO1 and transient receptor potential vanilloids (TRPVs), which transduce mechanical stimuli into intracellular signaling cascades and transcriptional programs^150–152. These mechanotransduction pathways determine EC fate and differentiation, thereby guiding vascular morphogenesis. For example, recent alveolus-on-a-chip model demonstrates that cyclic breathing-like deformations stimulate protective innate immune responses to viral infection in lung epithelial cells and ECs, suppressing viral replication through activation of TRPV4 and AGE/RAGE signaling¹⁵³. TRPV4 also mediates mitochondrial ROS-induced Ca²⁺ influx to promote abnormal EC migration and proliferation in an in vitro PAH model¹⁵⁴. PIEZO1 and PIEZO2, which exhibit an inverted dome shape structure, are reversibly flattened in response to mechanical stress^{155, 156}. In the lung, mechanical forces activate PIEZO1, triggering Ca²⁺ influx to regulates angiogenesis, vascular barrier function, and tissue repair across multiple cell types¹⁵⁷. PIEZO1 activation in neutrophils drives a lung-specific pro-angiogenic transcriptional program essential for capillary growth¹⁵⁸, whereas endothelial PIEZO1 senses alveolar stretch and microvascular pressure to disrupt EC junctional integrity and increases vascular permeability¹⁵⁷. PIEZO1 activation contributes to cardiopulmonary vascular diseases, including PH¹⁵⁹. Knockdown of Piezo1 in PASMCs reduces proliferation and contractility, thereby attenuating PH phenotype in a mouse model¹⁶⁰. In contrast, knockdown of Piezo2 in ECs exacerbates PA vasoconstriction in a rodent PH model¹⁶¹. These findings suggest that integration of mechanosensitive signaling pathways across multiple cell types governs lung vascular morphogenesis and function.

Age-dependent changes in ECM components and structures can alter EC response to mechanical stimuli and impair mechanotransduction. Altered compliance, vascular pressure, and ECM stiffness in the aged lung influence with each other to distort normal force distribution, generating an aberrant microenvironment that no longer supports angiogenesis. The resulting EC dysfunction leads to vascular rarefaction, impaired alveolar regeneration, and increased susceptibility to fibrotic or emphysematous remodeling. Several key mechanosensitive pathways are involved in the processes. For example, YAP/TAZ activity is reduced, while Rho signaling such as CDC42 activity is increased in aged ECs²¹. CDC42-YAP signaling responds to age-dependent increases in EC size, promoting EC senescence to inhibit angiogenesis in aged tissue²¹. Reducing aged EC size by culturing on single-cell sized smaller micropatterned islands decreases CDC42 activity, enhances YAP1 nuclear translocation, and attenuates EC senescence²¹. In addition, although expression levels are unchanged, reduced endothelial TRPV4 function leads to abnormal Ca²⁺ signaling and impaired vasodilation in aging mesenteric arteries¹⁶². Increased interaction between G protein-coupled receptor 35 (GPR35) and TRPV4 in aging ECs contributes to endothelial dysfunction and impairment of vasodilation¹⁶². Despite these findings, the direct interaction between aging and the response to mechanical forces in lung ECs has not been fully understood. Investigating the effects of aging on lung EC responses to mechanical forces would identify new targets to restore vascular morphogenesis and function in the aged lung.

IV. New models to study lung angiogenesis

Understanding the complex processes of lung angiogenesis requires diverse experimental models and methodological approaches that capture structural and functional dynamics of the pulmonary vasculature. Given the architectural complexity of the lung and substantial differences between human and animal lungs -including signaling pathways, anatomy, and physiology- conventional rodent models and traditional in vitro cell culture systems have significant limitations. Therefore, advanced technologies such as three-dimensional (3D) lung hydrogel implantation systems, organoid models, and organ-on-a-chip platforms, combined with organ-specific human cells, are warranted. These complementary approaches offer distinct advantages in recapitulating physiological and pathological microenvironments and preserving lung vascular structure and function.

Hydrogel implantation system

Hydrogel implantation assays, in which Matrigel is typically implanted subcutaneously, enable the investigation of EC sprouting, migration, and lumen formation in a 3D context. These assays are well suited for mechanistic studies and high-throughput screening of pro- and anti-angiogenic factors. However, because angiogenic responses and signaling pathways are organ specific, this method has been modified to implant the gel on the mouse lungs to study lung-specific angiogenesis¹⁶³. In this modified approach, hydrogels fabricated from different ECM components (e.g., collagen, fibrin) and supplemented with angiogenic factors are implanted on the lung surface of living mice. This enables the analysis of lung-specific angiogenesis and interactions with alveolar epithelial cells within the implanted gel, which cannot be achieved using conventional subcutaneous implantation. By modulating gel stiffness and ECM components, supplementing growth or angiogenic factors, and including specific cell types with targeted gene manipulations, this approach allows systematic investigation of the effects of chemical and mechanical cues on lung angiogenesis^{81, 163}. Implantation of hydrogels supplemented with human lung ECs with diverse conditions (e.g., age, race, sex, body mass index (BMI), disease status) to immunocompromised mouse lungs enables the study of human EC signaling and pathology⁸¹. This approach allows us to (i) clearly visualize EC morphogenesis and cellular interactions and (ii) delineate signaling pathways in the supplemented ECs. Compared with generating transgenic animals, this strategy is more time- and cost-efficient and reduces animal usage. Combination of hydrogel implantation with transgenic animal models will further efficiently and precisely elucidate the mechanisms of angiogenesis in the lung.

Organoid system

Organoids −3D tissue structures that potentially recapitulate cellular composition, cell-cell interactions, and key organ functions- are powerful platforms for studying human development and disease. Organoids derived from human pluripotent stem cells or tissue-resident progenitors are widely used to investigate cell fate specification and signaling pathways across multiple organs, including the lung^{164, 165}. However, there are challenges in current organoid systems; (i) lack of physiological perfusion that restricts oxygen and nutrient diffusion and limits long-term viability; (ii) immature vascular structure and limited functionality due to incomplete recapitulation of microenvironment.

Recently, co-differentiation of mesoderm and endoderm within the same spheroid has been shown to enable vascularization of lung and intestinal organoids derived from induced pluripotent stem cells (iPSCs) by tuning the endoderm-to-mesoderm ratio through bone morphogenetic protein (BMP) signaling¹⁶⁶. These lung organoids with organ-specific blood vessels exhibit enhanced cellular diversity, improved 3D architecture, and increased cell survival and maturation, which enables studying human organogenesis and disease mechanisms. It has also been demonstrated that microinjection of mouse lung ECs into bronchioalveolar lung organoids generates endothelialized organoids with integrated alveolar capillary networks¹⁶⁷. Upon exposure to inflammatory stimuli, such as LPS or influenza virus, these bronchioalveolar lung organoids recapitulate endothelial inflammatory responses¹⁶⁷. To directly investigate human EC morphogenesis and signaling, 3D human EC organoid system has also been developed, in which ECs freshly isolated from human tissues with varieties of conditions (e.g., age, sex, BMI, race, diseases) are cultured in the hydrogels in vitro¹⁶⁸. This approach allows (i) clear visualization of EC sprouting and enables (ii) analysis of EC signaling by manipulating expression of multiple genes.

ECs are highly heterogeneous, and therefore maintaining organ-specific EC identity in primary cultures and organoid systems remains challenging once cells are removed from their native microenvironment¹⁶⁹. Preserving EC identity—critical for reliable preclinical models—requires the reconstruction of organ-specific niches by incorporating key microenvironmental cues, including physiological nutrient and oxygen levels, flow, ECM components and stiffness, and interactions with resident cells^{170, 171}. Combination of human EC organoid systems with in vivo mouse disease models, including transgenic animal models, will enable more efficient and precise analysis of the mechanism underlying vascular and alveolar repair/regeneration in the lung and other organs. EC organoid systems across species will also facilitate cross-species comparisons to identify conserved and divergent mechanisms.

Organ-on-a-chip microfluidic system

Organ-on-a-chip microfluidic device – a platform containing hollow channels lined with organ-specific living cells cultured under fluid flow- is another promising approach to study human biology and disease pathology, as well as to evaluate drug toxicity and efficacy for future clinical translation. Among these chip platforms, the human lung-on-a-chip is the first organ-on-a-chip model developed¹⁷². It consists of two parallel microchannels separated by a microporous membrane; human ECs exposed to shear flow are cultured in one channel, while human lung alveolar epithelial cells under cyclic stretch are cultured in the opposing channel, replicating pulmonary vascular perfusion and breathing motions¹⁷². This platform has been further modified to model (i) inflammation-induced thrombosis by flowing human whole blood, (ii) COPD or cystic fibrosis by exposing cells to cigarette smoke or using patient-derived airway epithelial cells, and (iii) viral infection, including COVID-19 or influenza^173–175. To date, a wide range of human diseases and clinical responses across organs have been modeled using different organ-on-a-chip platforms engineered with specific cell types and stimuli¹⁷⁶. Further modification to fluidically connect multiple organ-on-a-chip platforms will enable understanding systemic processes, including hormonal effects, tumor metastasis, and drug pharmacokinetics, improving the assessment of drug toxicity and efficacy for lung and other diseases. Combination of organoid systems with organ-on-a-chip models by using patient-derived cells or iPSCs cultured/differentiated in the organoids for the organ-on-a-chip platform to apply physiological mechanical forces, perfusion and oxygen gradients is currently under development. This integrated approach has the potential to synergize the advantages of each system and overcome their limitations^{177, 178}.

Conclusions

Angiogenesis and EC signaling are critical for alveolar development and lung regeneration, while age-dependent decline in angiogenesis contributes to the pathogenesis of chronic lung diseases. Although aging alters numerous biological processes and molecular programs, how lung vasculature and EC signaling change with age and how these alterations impair alveolar morphogenesis and lung injury repair remain poorly understood. Future studies integrating single cell transcriptomics with spatial omics technologies will define the spatiotemporal dynamics of growth factors, ECM components, and EC subpopulations in the aged lung, advancing our understanding of the role of lung ECs in age-dependent decline in lung vascular and alveolar regeneration. Studies leveraging human lung ECs isolated from surgical specimens, adult lung stem cells, or human iPSCs-derived vascular cells, combined with emerging approaches such as organoid systems and organ-on-a-chip technologies, will facilitate the identification and validation of therapeutic targets with strong translational potential. For example, senolytic therapies have been demonstrated to attenuate tissue injury, extend lifespan, and delay age-associated pathologies, and their clinical applications are extensively explored¹⁷⁹. However, senolytic therapies are still not highly successful, partly due to associated side effects¹⁷⁹. Further understanding of how ECs maintain lung vascular and alveolar structures and homeostasis, and how these processes become dysregulated with aging using complementary models, including young vs. aged preclinical mouse models, human patient-derived tissues and ECs, lung organoid systems, and lung-on-a-chip platforms, will lead to more effective therapeutic strategies. These knowledges are critical for optimizing interventions, including senolytic therapies (e.g., dose, timing), to maximize therapeutic benefit for age-related lung diseases.

Highlights.

• Impaired endothelial signaling during aging leads to age-dependent decline in lung vascular and alveolar regeneration.

• Disruption of angiogenic signaling contributes to age-associated lung diseases.

• Age-associated changes in ECM component and structures compromise the micromechanical environment in the lung and inhibit lung vascular regeneration.

• New technologies, including organoid models and organ-on-a-chip platforms better recapitulate lung vascular and alveolar structure and function and may enable effective therapies for age-related lung diseases.

Non-standard Abbreviations and Acronyms:

ACKR1

atypical chemokine receptor 1

AGEs

advanced glycation end products

ALDH

aldehyde dehydrogenase

ALK1

activin receptor-like kinase 1

AMPK

AMP-activated protein kinase

ANGPT1

angiopoietin 1

APLN

apelin

APLNR

apelin receptor

AT2 cells

type II alveolar epithelial cells

ATF3

activating transcription factor 3

ATM

ataxia-telangiectasia mutated

ATR

ataxia-telangiectasia and Rad3-related

BMI

body mass index

BMP

bone morphogenetic protein

BPD

bronchopulmonary dysplasia

CAP1

capillary type 1 endothelial cell

CAP2

capillary type 2 endothelial cell

CDKN1A

cyclin dependent kinase inhibitor 1A

COPD

chronic obstructive pulmonary disease

CTGF

connective tissue growth factor

CXCL1

C-X-C motif chemokine ligand 1

3D

three-dimensional

DDR

DNA damage response

EC

endothelial cell

ECM

extracellular matrix

eNOS

nitric oxide synthase 3

ERG

ETS-related gene

FAK

focal adhesion kinase

FGF

fibroblast growth factor

FOXM1

forkhead box protein M1

FOXO1

forkhead box protein O1

FXYD5

FXYD domain containing ion transport regulator 5

GPR35

G protein-coupled receptor 35

ICAM1

Intercellular adhesion molecule 1

IFN-γ

Interferon-gamma

IGFBP7

insulin like growth factor binding protein 7

IL-6

interleukin-6

IPF

idiopathic pulmonary fibrosis

iPSCs

induced pluripotent stem cells

JNK

c-Jun N-terminal kinase

αKG

α-ketoglutarate

αKGM

α-ketoglutaramate

LMNA

lamin A/C

LOX

lysyl oxidase

LOXL

LOX-like enzyme

LPS

lipopolysaccharide

LRG1

leucine-rich α−2-glycoprotein 1

LRP5

low-density lipoprotein receptor-related protein 5

MAPK

mitogen-activated protein kinase

METTL3

methyltransferase-like 3

MMP14

matrix metalloproteinase 14

mTORC1

mechanistic target of rapamycin kinase C1

MUC1

mucin 1

NF-kappaB

nuclear factor kappa B

NIT2

nitrilase family member 2

NO

nitric oxide

NOX

NADPH oxidase

NTRK2

neurotrophic receptor tyrosine kinase 2

PA

pulmonary artery

PAH

pulmonary arterial hypertension

PAI-1

plasminogen activator inhibitor type 1

PASMCs

PA smooth muscle cells

PDGF-AA

platelet-derived growth factor AA

PECAM1

platelet and endothelial cell adhesion molecule 1

PGC1α

peroxisome proliferator-activated receptor gamma coactivator 1-alpha

PH

pulmonary hypertension

PI3K

phosphoinositide 3-kinase

PNX

pneumonectomy

PROCR

protein C receptor

RAGE

advanced glycosylation end-product specific receptor

ROS

reactive oxygen species

SASP

senescence-associated secretory phenotype

SA-β-gal

senescence-associated β-galactosidase

scRNA-seq

single-cell RNA sequencing

SENP1

SUMO-specific protease 1

SERPINE1

serpin family E member 1

SIRT3

sirtuin 3

SQLE

squalene epoxidase

SRF

serum response factor

TERC

telomerase RNA component

TERT

telomerase reverse transcriptase

TGF-β1

transforming growth factor beta 1

TIMP3

tissue inhibitor of metalloproteinases 3

TNF-α

tumor necrotic factor-alpha

TPI1

triosephosphate isomerase 1

TRF

telomeric repeat binding factor

TRKB

tyrosine receptor kinase B

TRPV

transient receptor potential vanilloid

TSP1

thrombospondin 1

VEGFA

vascular endothelial growth factor A

VEGFR1

vascular endothelial growth factor receptor 1

YAP1

yes-associated protein 1

ZO-1

zona occludens 1