ReviewHistorical Article

Endothelial metabolism in pulmonary vascular homeostasis and acute respiratory distress syndrome

Reece P. Stevens

Department of Physiology and Cell Biology, College of Medicine, University of South Alabama, Mobile, Alabama

Center for Lung Biology, College of Medicine, University of South Alabama, Mobile, Alabama

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Sunita S. Paudel

Department of Physiology and Cell Biology, College of Medicine, University of South Alabama, Mobile, Alabama

Center for Lung Biology, College of Medicine, University of South Alabama, Mobile, Alabama

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Santina C. Johnson

Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, Alabama

Department of Biomolecular Engineering, College of Medicine, University of South Alabama, Mobile, Alabama

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Troy Stevens

Department of Physiology and Cell Biology, College of Medicine, University of South Alabama, Mobile, Alabama

Center for Lung Biology, College of Medicine, University of South Alabama, Mobile, Alabama

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, and

Ji Young Lee

Department of Physiology and Cell Biology, College of Medicine, University of South Alabama, Mobile, Alabama

Department of Internal Medicine, College of Medicine, University of South Alabama, Mobile, Alabama

Division of Pulmonary and Critical Care Medicine, College of Medicine, University of South Alabama, Mobile, Alabama

Center for Lung Biology, College of Medicine, University of South Alabama, Mobile, Alabama

Correspondence: J. Y. Lee ([email protected]).

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Published Online:10 Aug 2021https://doi.org/10.1152/ajplung.00131.2021

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Abstract

Capillary endothelial cells possess a specialized metabolism necessary to adapt to the unique alveolar-capillary environment. Here, we highlight how endothelial metabolism preserves the integrity of the pulmonary circulation by controlling vascular permeability, defending against oxidative stress, facilitating rapid migration and angiogenesis in response to injury, and regulating the epigenetic landscape of endothelial cells. Recent reports on single-cell RNA-sequencing reveal subpopulations of pulmonary capillary endothelial cells with distinctive reparative capacities, which potentially offer new insight into their metabolic signature. Lastly, we discuss broad implications of pulmonary vascular metabolism on acute respiratory distress syndrome, touching on emerging findings of endotheliitis in coronavirus disease 2019 (COVID-19) lungs.

INTRODUCTION

Although the endothelium was once thought to be inert, it is now recognized as a highly metabolic and interconnected cell network. Metabolism is required to support the essential functions that are innate to the endothelium, including junctional apposition, which is dynamic in nature and restrictive to water, solutes, and macromolecules, through processes that rely on ATP (1–3). Moreover, the metabolic processes intrinsic to this cell type defend against oxidative stress and fuel rapid migration and localized angiogenesis in response to injury (4–11). Endothelial cells rely on compartmentalized aerobic glycolysis to generate ATP within discrete subcellular regions and are dependent on the mitochondria for production of metabolites involved in macromolecular synthesis, cell signaling, and epigenetic regulation (5, 12, 13).

Lower respiratory tract infections disrupt the alveolar-capillary membrane leading to exudative edema and refractory hypoxemia that is life-threatening (14), an effect commonly seen in coronavirus disease 2019 (COVID-19) pneumonia. How viral and bacterial pneumonias influence lung capillary endothelial metabolism, and how these cells fuel the migration and angiogenesis that is necessary for the repair process, are issues fundamental to the pandemic. Here, we provide a perspective of the key historical insights that led to our current understanding of lung capillary endothelial cell metabolism in health and disease, and we consider lapses in our knowledge that may serve as directions for future research.

THE DISCOVERY OF AEROBIC GLYCOLYSIS AS A MECHANISM TO SUPPORT ATP SYNTHESIS IN PULMONARY MICROVASCULAR ENDOTHELIAL CELLS

The pulmonary circulation comprises three major vascular segments, including the arteries, capillaries, and veins (15). Pulmonary arteries enter the lungs at the hilum and transverse through each lobe to the peripheral border, carrying deoxygenated blood to the capillaries (15). Pulmonary arterial endothelial cells (PAECs) line arteries and arterioles, which have lumens that are greater than 60 µm in their internal diameter (16). These arteries give rise to the pulmonary capillaries. The pulmonary capillaries are a vast network of microvessels that surround alveoli and form the alveolar-capillary membrane, a barrier thin enough to allow gas exchange yet separate the circulation from the outside environment (15, 16). The alveolar-capillary interface is a niche environment, home to dynamic gas change, and a diverse array of cell types. Pulmonary microvascular endothelial cells (PMVECs) are a major constituent of the alveolar-capillary membrane. These cells line the capillaries, and vessels less than 38 µm in internal diameter, they form a restrictive barrier, and they communicate with other cell types in the region (15). PMVECs are mostly quiescent, or nonproliferating, but can quickly switch to a promigratory and proliferative state when expanding or repairing the capillary network.

PMVECs in the quiescent or promigratory state generate most of their ATP through aerobic glycolysis, a bioenergetic pathway denoted as the conversion of glucose to lactate in the presence of oxygen (4, 17). Aerobic glycolysis can be further subdivided into glycolysis, the conversion of glucose to pyruvate, and fermentation, the conversion of pyruvate to lactate. Although aerobic glycolysis is studied today for its importance in endothelial metabolism, some of the earliest observations and experiments that shaped our fundamental understanding of glycolysis date back to the eighteen- and nineteen-hundreds.

The History of Aerobic Glycolysis

In 1856, industrialist Monsieur Bigo faced economic distress when some of his fermentation containers at the distillery started converting beat sugar into a sour substance instead of alcohol (18–20). Bigo sought the help of chemist Louis Pasteur, hoping he could resolve the underlying cause. Upon analyzing the chemical composition of the sour substance, Pasteur found a high amount of lactate present. This was an unexpected discovery at the time since alcohol fermentation was believed to occur spontaneously from sugar. When he examined the sour substance under a microscope, he noticed it was contaminated with rod-like microbes (18–21). Pasteur’s astute observation led him to the realization that fermentation was not a spontaneous process but required the presence of microbes. He also noted that bacteria execute a different type of fermentation than yeast, producing lactate and acidic compounds instead of alcohol (18–21). After saving Bigo’s business, Pasteur continued to study alcoholic fermentation in yeast. In one of his most influential papers, Pasteur discovered that yeast lose the ability to ferment in the presence of oxygen but regain fermentation and rapid sugar consumption when oxygen is removed. Pasteur observed that yeast growth was directly linked to fermentation, and thus termed it “life without air” (18, 19, 21). Pasteur was the first to suggest fermentation is an essential chemical pathway in living cells and is strictly an anaerobic process.

By the start of the nineteen-hundreds, glycolysis was primarily studied for its role in microbial fermentation but was beginning to receive recognition for its importance in animals. In 1906, the British physiologist Walter Fletcher studied acid production in frog legs during contraction. He noticed that when muscle contracted under anaerobic conditions, acid and lactate were generated, but when muscle contraction occurred in the presence of pure oxygen, acid and lactate production was suppressed (22). Although little was known about the mechanisms of glycolysis at the time, Fletcher noted the process was similar to fermentation in yeast (22).

The mechanisms of glycolysis in cellular energetics continued to unravel throughout the early 1900s. Otto Meyerhof, a German physician and biochemist, studied the mechanism of glycolysis, discovering over one-third of the glycolytic enzymes, and in 1918, demonstrated that yeast and animal cells share glycolytic coenzymes, directly connecting the biochemical pathway from microbes to animals (23–25). Meyerhof concentrated his work on glycolysis in the muscle of frog legs. Like Fletcher, Meyerhof noticed lactate only appeared in contracting muscle under anaerobic conditions. He was able to expand upon Fletcher’s studies, demonstrating lactate gradually appears in equivalent quantities to the loss of glycogen, thus proving the conversion of glycogen to lactate as an energy source in muscle (23–26). Meyerhof’s and Fletcher’s findings in muscle metabolism reinforced the principle that glycolysis is strictly an anerobic process and was thought to occur in muscle only when the energy demand outpaces energy received from cellular respiration (27). Thus, the conversion of glucose to lactate in animals became known as anaerobic glycolysis.

In 1922, Meyerhof was awarded a Nobel Prize for his work in muscle metabolism (28). During this same year, however, Otto Warburg discovered an exception to anerobic glycolysis that would become the key to our modern day understanding of how capillaries sustain their energetic demands. Warburg was raised in an academic-rich environment due to his father’s status as an eminent professor in physics, frequently having guests like Albert Einstein and Max Planck over for social events (28). During the start of World War I, Warburg enlisted into the German military as a physician, but eventually left the army after receiving a letter from Einstein (at the request of Warburg’s mother), pleading with Warburg to return to the laboratory and continue his scientific career (28). Back at the laboratory, Warburg’s exposure to prominent physicists proved useful as he learned how to repurpose a manometer to measure CO₂ production and O₂ consumption in metabolic studies of cells and tissue (28). Warburg was studying cancer metabolism when he observed that the addition of glucose to tumors led to a color change in the Ringer solution. The color change was based on an organic pH indicator, suggesting the solution had acidified. Upon chemical analysis of the solution, Warburg identified high amounts of lactate present (28, 29). Following this study, Warburg examined hepatoma tissue from rats. He found the hepatoma tissue formed 70 times more lactate than normal tissue (28, 29), and in vivo, had increased lactate in blood vessels leaving tumors compared with blood vessels entering tumors (28, 30) (Table 1). The production of lactate in oxygenated tissue suggested that glycolysis could be an aerobic or anaerobic process in a context-dependent manner. Many scientists, including Warburg himself, were perplexed by this discovery. Although the mechanism of cellular respiration was unknown at the time, Warburg understood glycolysis to be less energetically efficient than cellular respiration (31) and it remained a mystery as to how aerobic glycolysis could sustain tumor growth. Warburg attributed the phenomenon to damaged “gana” (mitochondria) in tumors, forcing the cell to rely on anaerobic glycolysis instead of cellular respiration for energy production (28, 31); an enticing idea at the time but now accepted to not be true in most cancers (32, 33). Warburg’s studies had three significant contributions to our understanding of metabolism: 1) glycolysis can occur in the presence of oxygen, referred to as aerobic glycolysis or the Warburg effect; 2) aerobic glycolysis can support a highly proliferative and metastatic cellular phenotype; and 3) cells that utilize aerobic glycolysis as their predominant bioenergetic pathway are highly acidifying. Warburg’s research focused on aerobic glycolysis in tumors, but cancer cells are not the only cell types that employ aerobic glycolysis.

**Table 1. Arterial and venous blood glucose and lactate measurements from rat tissues and Jensen sarcoma**
	Glucose in 100 mL of Blood
	Artery, mg	Vein, mg	Artery – Vein (Difference)	(Artery – Vein/Artery) × 100, %
Jugularis	99	97	+2	2
Renalis	111	108	+3	3
Iliaca	143	125	+18	13
Portae	91	75	+16	18
Jensen Sarcoma	124	54	+70	57
	Lactate in 100 mL of Blood
	Artery, mg	Vein, mg	Artery – Vein (Difference)
Jugularis	16	17	+1
Renalis	28	15	−13
Iliaca	44	39	−5
Portae	22	21	−1
Placenta	17	13	−4
Jensen Sarcoma	32	78	+46

Warburg discovers Jensen sarcoma has elevated aerobic glycolysis compared with normal rat tissue. Arterial blood was collected from the abdominal aorta and tumor arteries and venous blood was collected from the jugular, iliac, renal, portal, placenta, and tumor veins. Warburg’s blood and lactate measurements from artery and venous blood show Jensen sarcoma to have significantly higher glucose consumption and lactate production than normal rat tissue. [Adapted from Warburg et al. (30).].

Aerobic Glycolysis in the Pulmonary Endothelium

Endothelial cells also utilize aerobic glycolysis to meet their bioenergetic demand in the quiescent and promigratory state (1–3, 5–7, 34–40), however, this was far from the prevailing view during the 1960s. At that time, no method existed to study the endothelium in isolation, and physiological processes could only be inferred through electron microscopy imaging, leading scientists to believe that it acted as an inert barrier (41–43). In Lord Adrian Florey’s 1966 paper “The Endothelial Cell”, he described the endothelium as a “sheet of nucleated cellophane” (43). The idea that the pulmonary endothelial layer might have higher basal metabolic activity did not begin to surface until it was recognized between 1965 and 1970 that compounds including angiotensin I, serotonin, bradykinin, and prostaglandin were inactivated after moving through the pulmonary circulation (44–48). These studies hinted at the possibility that endothelial cells have active metabolic functions outside of their passive role as conduits for blood flow.

During this same time, physicians Ralph Nachman and Eric Jaffe were studying hemostasis and had developed an unconventional idea that the endothelium was involved in regulating blood coagulation. Validating their idea proved challenging as they had no approach to study the endothelium directly, since no endothelial isolation and culture method existed (41). To overcome this barrier, Nachman and Jaffe decided to develop a method for endothelial culture, a decision which received much skepticism because their laboratory had no experience in tissue culture and previous attempts to culture the endothelium by other experts proved unsuccessful (41). Jaffe, a hematology fellow at the time, consulted with graduate student Karen Artz who had expertise in cell culture. After receiving a brief introduction to cell culture and reflecting on the available literature, Jaffe began his first attempt to isolate and culture the endothelium from a human umbilical vein (42). He made a crucial decision. Jaffe changed some of the steps in the experimental protocols used in previous attempts to isolate the endothelium. He changed the digestive enzyme from trypsin to collagenase and increased the amount of fetal calf serum in the media (42). When Jaffe returned to check on the isolated cells the next day, he found a monolayer of flat, polygonal-shaped cells (42, 49) (Fig. 1A).

**Figure 1.**Endothelial cell culture. A: a photomicrograph of the first reported endothelial cell culture. [Republished with permission of the American Society for Clinical Investigation, from Jaffe et al. (49); permission conveyed through Copyright Clearance Center, Inc.] B: human umbilical vein endothelial cells with immunofluorescent staining against factor VIII. [Republished with permission of the American Society for Clinical Investigation, from Jaffe et al. (50); permission conveyed through Copyright Clearance Center, Inc.] C: confluent rat pulmonary arterial endothelial cells. D: the first pulmonary microvascular endothelial cell (PMVEC) culture isolated from rabbits, ×200. [Reprinted from Ryan et al. (52) with permission from Elsevier.]

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Nachman and Jaffe realized the acceptance of their discovery depended on verifying the purity of their culture, so in 1973, they collaborated with Lee Hoyer. Hoyer had previously demonstrated endothelial cells exclusively express von Willebrand factor, allowing Nachman and Jaffe to use it as an antigen marker to validate the purity of their cell culture (41, 42, 50) (Fig. 1B). Endothelial cell culture was a transformative technique in the 1970s and 1980s, and the success of Nachman and Jaffe was shortly followed by others, expanding endothelial cell culture from umbilical veins to other vascular beds. Una and James Ryan were the first to isolate and culture endothelial cells from pulmonary arteries and capillaries in 1978 and 1982, respectively (51, 52) (Fig. 1, C and D).

The concept of endothelial heterogeneity, or distinct functions and structures between endothelial cells from separate vascular beds, was a growing area of interest in the 1990s and 2000s. The ability to separately culture PAECs and PMVECs provided an approach for vascular biologists to rigorously study differences in endothelial phenotypes along the pulmonary arterial-capillary-venous axis. Several studies in the 1990s demonstrated PAECs and PMVECs to have distinct lectin-binding properties, allowing accurate discrimination between the two endothelial types (53–61). In 2003, King et al. (61) noted PMVECs grew twice as fast as PAECs in log phase growth, when cultured in the same conditions. PMVECs were even able to maintain rapid growth in limited serum if they were preconditioned in a serum-rich environment, whereas PAEC growth was inhibited by restricted serum. King’s work was followed by Alvarez and colleagues who examined the proliferative diversity within PMVEC and PAEC populations. Human aortic endothelial cells were previously shown to contain a hierarchy of endothelial progenitor cells, resulting in focal regions of rapid cell growth within the endothelial layer of the aortic wall (62, 63). To determine whether pulmonary vessels displayed a similar growth pattern, these investigators seeded PMVECs and PAECs at the single-cell level and measured growth 2 wk later. Over 50% of single cells derived from PMVEC populations displayed large colony growth, whereas less than 20% of single cells derived from PAEC populations grew into large colonies. Not only did PMVECs have a higher percentage of large colonies than PAECs, the large colonies were more populated (64). Large colonies derived from PMVECs consisted of 100,000 cells compared with the 10,000 cell colonies formed from PAECs (64). They next measured the capacity for PMVECs and PAECs to form blood vessels in vivo. PMVECs or PAECs were seeded in Matrigel and then subcutaneously injected. As predicted from the growth studies, PMVECs formed twice as many blood vessels than PAECs (64). The vessel lumen diameter varied between the two cell types, with PMVECs forming small vessels (<10 µm lumen) and PAECs forming larger vessels (<50 µm lumen) (64), indicating vessel lumen size is part of an intrinsic memory in endothelial cells. Alvarez et al.’s studies revealed PMVECs are enriched with progenitor cells, exhibiting rapid growth and angiogenesis, in part due to the presence of replication-competent cells within the cell population. This work demonstrated PMVECs have a unique proproliferative and angiogenic phenotype that is not seen in PAECs.

Rapid proliferation and angiogenesis are metabolically demanding characteristics, yet how PMVECs meet the bioenergetic demand to sustain growth was unknown in the early 2000s. Endothelial cells derived from the rat coronary, pig aorta, and bovine phallus showed a dependency on glycolysis for energy production (65–68). In addition, endothelial cells in general have few mitochondria (12) and are able to maintain ATP levels under low-oxygen conditions (69, 70), indicating PMVECs may not rely on cellular respiration as their major bioenergetic pathway. In 2010, Para-Bonilla et al. (4) sought to determine whether PMVECs utilize aerobic glycolysis as their primary energy source. She found that PMVECs rapidly convert glucose to lactate during growth, indicating they rely on aerobic glycolysis. Since lactate dehydrogenase (LDH) is a major enzyme that converts pyruvate to lactate in aerobic glycolysis, Para-Bonilla et al. investigated whether genetic ablation of LDH inhibits PMVEC growth. Not surprisingly, the proproliferative phenotype was inhibited by decreasing LDH expression (4). The investigators next studied whether aerobic glycolysis was necessary for angiogenesis in vivo using a Matrigel plug assay. LDH expressing PMVECs formed significantly more blood vessels compared with cells without LDH expression (8). Although Para-Bonilla et al. clearly demonstrated PMVECs depend on aerobic glycolysis, PAECs did not have the same dependency. PAECs showed little glucose consumption, lactate production, and LDH expression while having highly polarized mitochondrial membranes and twice the oxygen consumption compared with PMVECs, indicating that by comparison PAECs use cellular respiration to meet their energetic demands (4). Thus, Para-Bonilla’s work revealed that PMVECs and PAECs have distinct bioenergetic systems that drive different rates in proliferation and angiogenesis. Growth and angiogenesis were not the only phenotypic differences between these two energy systems, however. Similar to Warburg’s pH study on cancer, Para-Bonilla et al. (4) added glucose to cultured PMVECs and PAECs, and PMVEC growth was paralleled with acidosis of the culture media while rat PAEC media pH did not change. Therefore, it seems aerobic glycolysis provides PMVECs with a proproliferative and angiogenic phenotype, but at the cost of excess proton production, leaving us with a perplexing question; if PMVEC metabolism causes metabolic acidosis, how does it maintain pH homeostasis when excess proton generation is not conducive for growth and survival?

In order for PMVECs to handle the acid load generated during metabolism, PMVECs must have a high degree of acid tolerance. PMVECs maintain growth in pH as low as 6.2, whereas PAECs growth is stunted, indicating PMVECs have a higher proton regulatory capacity than PAECs (71). Endothelial cells across various vascular beds express Na⁺/H⁺-exchangers and ${HCO}_{3}^{-}$ transporting proteins as regulators of intracellular pH (72–77), so it seemed possible for there to be unique pH regulatory proteins increasing the acid tolerance of PMVECs. In 2004, Rojas et al. (78) found PMVECs, but not PAECs, express vacuolar-type H⁺-ATPase (V-H⁺-ATPase) in the plasma membrane, providing an additional mechanism to regulate intracellular pH independent of Na⁺ and ${HCO}_{3}^{-}$ . V-H⁺-ATPase was integral to intracellular pH maintenance in PMVECs, promoting proton efflux from the cell during intracellular acidification (78).

V-H⁺-ATPase is not the only distinct pH regulatory protein providing PMVECs with an enhanced proton regulatory capacity. PMVECs also express carbonic anhydrase IX (CA IX) (71), the most catalytically active carbonic anhydrase isoform typically expressed by aggressive forms of cancer and the gastric epithelium (79). Genetic ablation of CA IX inhibited PMVEC cell migration and angiogenesis under acidosis conditions, revealing CA IX as another major pH regulatory protein (71, 80). Thus, PMVECs exhibit enhanced proton buffering when compared with PAECs, due to the increased expression of unique pH regulatory proteins, which allow PMVECs to handle an excessive proton load produced by aerobic glycolysis.

Today, we understand that PMVECs utilize aerobic glycolysis to support their proproliferative and angiogenic phenotypes at the cost of excess proton production, requiring PMVECs to express unique pH regulatory proteins to preserve intracellular pH. This phenotype is much different than PAECs, which exhibit a greater reliance on oxidative phosphorylation for energy production and display low proliferation, angiogenesis, and proton regulation in vitro (4). The reasons why PMVECs and PAECs evolved distinct metabolic and pH regulatory systems have yet to be determined, but are likely to be a mixture of functional and environmental differences.

It is important to note these studies centered on PMVEC and PAEC proliferation, migration, angiogenesis, metabolism, and pH regulation were conducted in an in vitro setting. Cell culture is an informative approach but cannot replicate the complexities of an in vivo environment. Shear stress, matrix stiffness, cell-cell communication, and external signaling molecules alter the metabolic state of cells (81–85), thus requiring complementary in vivo approaches to unravel the physiological and clinical significance of endothelial metabolism.

Recently, several groups used transgenic mice with a Cre-Lox system to conditionally knockout key metabolic enzymes from the endothelium, or alternatively, use pharmacological/molecular inhibitors to suppress metabolism pathways in vivo (1–3, 5–7, 34–37). Glycolytic proteins hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2) are rate-limiting enzymes (1, 6, 36), whereas 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is a potent allosteric activator (5, 86, 87), making them natural targets to manipulate glycolysis. Endothelial-specific deletion of these enzymes reveals aerobic glycolysis to have essential in vivo functions in both quiescent and promigratory endothelial cells (1–3, 5–7, 34–37). Similar approaches also demonstrate fatty acid oxidation (FAO) and other metabolic pathways to have equally important and complementary functions in the endothelium (10, 88, 89). Together, these studies illustrate how endothelial metabolism maintains pulmonary vascular integrity in vivo.

ENDOTHELIAL METABOLISM PRESERVES PULMONARY VASCULAR HOMEOSTASIS

Metabolism is a highly adaptable system which changes in response to external stress, nutrients availability, and cell activity (90). This flexibility is critical for cellular homeostasis and requires coordination between metabolic pathways to perform complementary functions and fulfill cellular demands (90). Glucose oxidation and FAO are two pathways that regulate each other’s activity (90, 91). As one oxidative pathway is upregulated, the other is suppressed (90, 91). The reciprocal relationship between glucose oxidation and FAO is known as the Randle cycle (90, 91). The Randle cycle is not limited to FAO, however, as glucose oxidation and glutaminolysis also have a reciprocal relationship (90). In line with the Randle cycle, endothelial cells exhibit high FAO (10, 88) and glutaminolysis (92, 93) while having little glucose oxidation (4). Instead of glucose oxidation, endothelial cells rely heavily on aerobic glycolysis for ATP production (4) and utilize FAO and glutaminolysis in the mitochondria to synthesize metabolites involved in biomass production (88, 93), antioxidant defense (10), and cell signaling (94, 95). This metabolic organization is vital to endothelial function and preserves pulmonary vascular homeostasis in five main ways: 1) regulation of endothelial permeability, 2) antioxidant defense, 3) pulmonary vascular repair, 4) postnatal angiogenesis, and 5) metabolite signaling and epigenetic regulation.

Aerobic Glycolysis Regulates Pulmonary Vascular Permeability

Permeability of the endothelial barrier is importantly determined by the presence of vascular endothelial cadherins (VE-cadherins), which tether neighboring endothelial cells at adherens junctions (AJs) (96–98). VE-cadherin tethering is coupled with intracellular contractile forces (mediated by the actin cytoskeleton) to generate tension at the cell-cell junctions (97, 98). A balance between cell-cell tethering and junctional tension is required to stabilize AJs and maintain a restrictive barrier (97). Hence, the strength of the endothelial barrier is regulated through actin remodeling and VE-cadherin internalization/trafficking to AJs (96–98), both of which are energetically taxing processes.

To meet this bioenergetic demand, glycolytic enzymes compartmentalize to the membrane and supply localized pools of ATP for actin rearrangement and VE-cadherin vesicle trafficking (1–3) (Fig. 2A). The rate of glycolysis and subsequent ATP production at AJs is an important factor in regulating barrier permeability. Endothelial-specific PKM2 knockout mice have high basal pulmonary microvascular permeability and edema (6). Silencing PKM2 in mice lung endothelial cells depletes the ATP content at AJs, disrupting normal VE-cadherin recycling and causing discontinuous junctions between cells (1). These PKM2 studies support the notion that a minimum amount of ATP is required at AJs to sustain a restrictive endothelial monolayer.

**Figure 2.**Endothelial metabolism regulates pulmonary vascular permeability and defends against oxidative stress. A: 1) glycolysis generates ATP for vascular endothelial (VE)-cadherin internalization and membrane trafficking to adherens junctions (AJs), controlling vascular barrier integrity (1–3). 2) Glycolytic enzyme pyruvate kinase M2 (PKM2) suppresses NF-κB signaling whereas 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activates NF-κB, regulating the inflammatory response and release of vascular permeabilizing agents (2, 6, 34, 103). 3) Fatty acid oxidation (FAO) is critical for redox homeostasis in quiescent endothelial cells, producing reduced nicotinamide dinucleotide phosphate (NADPH) to quench reactive oxygen species (ROS) and prevent oxidative damage to the endothelial barrier (10). B: the rate of glycolysis and subsequent ATP production regulates endothelial barrier permeability. High levels of compartmentalized ATP at AJs favor VE-cadherin internalization and activate NF-κB, permeabilizing the endothelial barrier (1–3, 103). Depleted ATP content at the membrane impairs AJ dynamics and actin remodeling, disrupting normal barrier homeostasis (1, 99). Optimal ATP content limits VE-cadherin internalization while retaining enough ATP to support AJ dynamics and actin remodeling, stabilizing the endothelial barrier. [Images created with Biorender.com and published with permission.]

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Chemical inhibition of PFKFB3 with 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) destabilized AJs in human PMVECs by disrupting VE-cadherin recycling and global actin networks, increasing cell-cell gap formation and compromising the endothelial barrier (99). In opposition to this, treatment of human umbilical venous endothelial cells (HUVECs) with the PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) blocked VE-cadherin endocytosis, leading to a tighter barrier with fewer gaps (2). These contrasting results could be cell type-specific but are more likely related to the chemical inhibitors used. 3PO is a weak competitive inhibitor whereas PFK15 is a more potent derivative (100). Even in the quiescent state, retaining some level of AJ dynamics is important for barrier homeostasis (101, 102). PFK15 might inhibit glycolysis too strongly, depleting the ATP content below the minimum amount needed to sustain basal barrier homeostasis, whereas P3O might lower the ATP content enough to reduce VE-cadherin internalization but not cause an ATP deficiency. Another possible scenario is different off target effects between PFK15 and 3PO. These are important issues that will need to be better resolved in the future.

When in the promigratory state, endothelial cells upregulate PFKFB3 to increase ATP and lactate production (2, 3, 5, 7, 37). The dramatic elevation in ATP content fuels VE-cadherin internalization, removing cell-cell tethering in favor of migration and angiogenesis (1, 3) (Fig. 2, A and B). Therefore, it seems the rate of compartmentalized glycolysis at AJs closely regulates vascular permeability with depleted and excessive ATP levels favoring permeability whereas basal ATP levels stabilize the endothelial barrier (Fig. 2B).

In addition to regulating permeability through ATP production, PFKFB3 and PKM2 have opposing regulatory roles in NF-κB signaling (Fig. 2A). Upregulation of PFKFB3 increases intracellular lactate levels which subsequently activates NF-κB, leading to higher endothelial permeability (2, 103). PKM2, on the other hand, suppresses NF-κB signaling, preventing the release of angiopoietin 2, a potent vascular permeability factor (6, 34). Thus, increased expression of PFKFB3 elevates the ATP content at AJs and induces NF-κB signaling to favor endothelial permeability, whereas PKM2 maintains normal ATP levels at AJs and suppresses NF-κB signaling to preserve the endothelial barrier (Fig. 2, A and B).

Endothelial Fatty Acid Oxidation as an Antioxidant Defense

The alveolar-capillary interface is an oxygen-rich environment, making PMVECs susceptible to oxidative stress. Reactive oxygen species (ROS) disrupt the vascular barrier if not quenched, requiring endothelial cells to have antioxidant defenses (10, 96). In particular, fatty acid oxidation (FAO) is increased by three- to fourfold in quiescent HUVECs to sustain redox homeostasis (10) (Fig. 2A). Quiescent HUVECs depend on FAO to generate a carbon source that is shuttled into the TCA cycle (10). In the TCA cycle, the carbon source regenerates reduced nicotinamide adenine dinucleotide phosphate (NADPH) which is subsequently used as a substrate by glutathione reductase to produce the potent antioxidant glutathione (GSH) (10). In addition, nicotinamide adenine dinucleotide (NAD⁺) salvaging enzymes are upregulated by quiescent HUVECs (10), likely contributing to the high NADPH levels. Sustaining NADPH levels in quiescent endothelial cells are necessary for quenching ROS. Knockdown of carnitine palmitoyltransferase 1A (CPT1A), the rate controlling enzyme in FAO, in quiescent endothelial cells show lower quantities of NADPH and a subsequent increase in ROS levels (10). To compensate for loss of FAO, isolated endothelial cells from endothelial-specific CPT1A knockout mice upregulate other pathways involved in redox homeostasis, including glutathione metabolism, ROS scavenging enzymes, and the pentose phosphate pathway (10). These compensatory mechanisms improve redox homeostasis but cannot safeguard endothelial integrity during ROS stress. This is evident as endothelial-specific CPT1A knockout mice have increased vascular dysfunction and permeability from inflammation-induced oxidative stress, especially in the lungs (10). The increased vascular damage was rescued following antioxidant treatment (10). Overall, these studies indicate quiescent endothelial cells reprogram their metabolism to defend against oxidative stress, maintaining vascular integrity in an oxygen-rich environment and during inflammation.

Endothelial Glycolysis Facilitates Pulmonary Vascular Repair and Lung Regeneration

Endothelial cells remain quiescent for years but can rapidly transition into a promigratory and proliferative state (38, 39, 104). Therefore, these cells are considered to have a high degree of plasticity that allows a swift response to environmental changes and stimuli (38, 39, 104). Disruption of the lung endothelial monolayer initiates this reparative state and is vital to restoring the alveolar-capillary membrane after injury (105). The promigratory and proliferative phenotype is largely driven by the upregulation of aerobic glycolysis (5). Advancements over the past several years have brought mechanistic insight into how aerobic glycolysis facilitates endothelial migration, proliferation, and angiogenesis.

During migration, the actin cytoskeleton remodels to form filopodia and lamellipodia at the leading edge of cells, requiring large quantities of ATP to fuel cytoskeletal rearrangements. Filopodia and lamellipodia are too thin to contain mitochondria, and mitochondria are not a reliable source of ATP for endothelial migration, since inhibiting the mitochondria in vitro does not impair migration (5). Instead, De Bock et al. (5) found glycolytic enzymes compartmentalize at the filopodia and lamellipodia. PFKFB3, pyruvate kinase, and other glycolytic enzymes bind to F-actin in these cellular regions, forming a glycolytic hub that generates a local supply of ATP for remodeling of the actin cytoskeleton (5, 13) (Fig. 3A). The importance of these glycolytic hubs is evident, as endothelial-specific deletion of PFKFB3 and PKM2 impairs vascular migration in vitro and in vivo (2, 3, 5–7, 34, 37). Interestingly, expression of active glycolytic enzymes that cannot colocalize with sarcomeres in Drosophila flight muscles results in fruit flies that cannot fly, further highlighting the significance of glycolytic compartmentalization in cell function (106).

**Figure 3.**Aerobic glycolysis and metabolite signaling support endothelial cell proliferation and migration for vascular repair and sprouting angiogenesis. Disruption of the endothelial barrier leads to alveolar edema. After injury, pulmonary microvascular endothelial cells (PMVECs) transition into a promigratory and proliferative state to rapidly repair the barrier (38, 39, 104). This reparative phase is dependent on an increase in aerobic glycolysis to sustain the metabolic demands of migration and proliferation (5). A: during endothelial migration, glycolytic enzymes colocalize with F-actin at the filipodia and lamellipodia, supplying a local pool of ATP for cytoskeletal rearrangement in migrating endothelial cells (5, 13). pH regulatory proteins line the filipodia and lamellipodia membrane to remove generated protons and maintain optimal intracellular pH for glycolytic enzymes and cytoskeletal proteins (108–111). A pH gradient forms along the longitudinal axis of the cell with a more alkaline pH at the leading edge and increasing acidity toward the cell body (108–111). B: during proliferation, the majority of glucose is converted to lactate for ATP production, but some glycolytic intermediates are shuttled to side pathways and the TCA cycle for the synthesis of amino acids and nucleotides (93). Intermediate metabolites and glycolytic enzymes also regulate cell cycle progression (6, 7, 34, 121, 122), likely coordinating the synthesis of ATP and macromolecules with specific phases of the cell cycle. C: sprouting angiogenesis is the formation of a new blood vessel that branches from an existing vessel (129). Glycolysis supports sprouting angiogenesis by supplying ATP for tip cell migration and stalk cell proliferation (1–3, 5–7, 34–37). Intussusceptive angiogenesis is the splitting one blood vessel into two separate vessels (126–129). Sprouting and intussusceptive angiogenesis are necessary during alveolarization for proper lung development and retention of capillary density in tissue (137, 138, 141). D: metabolites from the TCA cycle regulate the epigenetic landscape of endothelial cells and have extracellular signaling functions (94, 95, 150). α-Ketoglutarate (α-KG) dependent dioxygenase enzymes are key demethylases and hydroxylases that suppress hypoxia-inducible factor (HIF)-1 expression and control the methylation status of DNA (94, 95). The catalytic reactions of these enzymes require α-ketoglutarate but are impaired by succinate and fumarate (94, 95). The balance between α-ketoglutarate and succinate/fumarate regulates the activity of α-KG-dependent dioxygenase enzymes and the subsequent stability of HIF-1 and methylation status of DNA (94, 95). Accumulation of intracellular succinate/fumarate activates HIF-1 and initiates angiogenesis in aortic endothelial cells (148). The activity of acetylases and sirtuin deacetylases depends on the bioavailability of acetyl-CoA and NAD⁺ respectively (95, 144). Increasing endothelial NAD⁺ activates sirtuin 1, improving the angiogenic potential of endothelial cells (144). In the plasma, succinate acts as an extracellular ligand for the succinate receptor 1 (SUCNR1) in kidney and aortic endothelial cells, stimulating the renin-aldosterone-angiotensin system (RAAS) (150) and enhancing angiogenesis (148). [Images created with Biorender.com.] CA IX, carbonic anhydrase IX; MCT, monocarboxylate transporter; Na⁺/H⁺ exchanger, sodium-proton exchanger; V-H⁺-ATPase, vacuolar-type H⁺-ATPase.

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ATP hydrolysis is a major source of protons (107). The rapid ATP turnover in filopodia and lamellipodia will create an acidic environment that is suboptimal for enzyme activity and actin trailing if protons are not extruded at a high rate. To ensure intracellular acidosis does not occur, pH regulatory proteins are strategically positioned at the leading-edge membrane of migrating cells to remove and neutralize excess proton and lactate production (Fig. 3A). The V-H⁺-ATPase, Na⁺/H⁺ exchanger, and CA IX localize at the leading edge membrane of cardiac MVECs, murine fibroblasts, and Madin-Darby Canine Kidney (MDCK) cells, respectively (108–110). Subsequently, a pH gradient forms inside the cell with an alkaline pH at the leading edge and a gradual decline in pH along the longitudinal axis of the cell toward the lagging edge (108–111) (Fig. 3A). The alkaline pH at the leading edge likely sustains optimal pH for glycolytic enzyme function and cytoskeleton remodeling. Chemical and/or genetic inhibition of V-H⁺-ATPase and CA IX suppresses MVEC wound healing in vitro (80, 108, 112), suggesting that the alignment of pH regulatory proteins in the membrane is imperative for maintaining optimal intracellular conditions during migration. It is important to note, however, that these studies were limited to in vitro conditions and future in vivo experiments are needed to fully determine the role of pH regulatory proteins in vascular migration.

Aerobic glycolysis supports endothelial proliferation in three ways: ATP generation, carbon biomass production, and cell cycle regulation (2, 5–7, 34, 36–39, 104, 113, 113, 114) (Fig. 3B). Endothelial cells derive up to 85% of their ATP from aerobic glycolysis and are necessary to meet the bioenergetic demand of proliferation (4, 5). It is estimated to be 100 times faster than oxidative phosphorylation, allowing higher ATP generation in a short period despite its lower ATP yield (115–117). The fast kinetics of aerobic glycolysis was initially postulated to be most advantageous to biomass production, quickly producing glycolytic intermediates for synthesis of macromolecules (113). This notion has been challenged, however, as Kim and coworkers discovered HUVECs convert 90% of glucose into lactate, only leaving 10% for the synthesis of macromolecules, and thus, is not a bulk carbon source (93). Nevertheless, the small quantities of glucose used for nucleotide and amino acid production are still critical for endothelial proliferation as impairment of biosynthetic pathways branching from glycolysis cause vascular defects in vivo (2, 39, 118, 119).

The bulk source of biomass in the endothelium is now recognized to come from glutamine and fatty acids (88, 89, 93, 120). Glutamine metabolism replenishes TCA cycle intermediates whereas fatty acid oxidation provides a carbon source for nucleotide synthesis during proliferation in HUVECs (88, 89, 93, 120). Inhibition of glutamine and fatty acid metabolism in vivo suppresses endothelial proliferation without causing an energy deficiency (88, 89, 93, 120). Thus, glycolysis’ contribution to biomass production in the endothelium is less paramount than initially believed, but it still has a role to play in the synthesis of macromolecules (2, 39, 93, 118).

Recent discoveries found glycolytic enzymes regulate the cell cycle (6, 7, 34, 121, 122). This new function may coordinate energy production and macromolecule synthesis with specific phases of the cell cycle. Knockdown of PFKFB3 in HUVECs inhibits G1/S transition (7). Studies in HeLa cells demonstrated PFKFB3 traffic to the nucleus where it produces fructose 2,6-bisphosphate, which activates cyclin-dependent kinase-1 (Cdk1). Cdk1 then phosphorylates p27, resulting in p27 degradation and the G1/S transition (121, 122). Whether PFKFB3 uses the same mechanism to regulate the cell cycle in the pulmonary endothelium has yet to be determined, but it certainly has a role in promoting endothelial cell cycle progression. PKM2 may be another cell cycle regulator, as silencing of PKM2 in HUVECs led to cell cycle arrest and knockout of PKM2 inhibits endothelial proliferation in vivo (6, 34). Interestingly, the noncatalytically active dimeric form of PKM2 promotes cell cycle progression by suppressing p53, dissociating PKM2’s kinase activity from its function as a cell cycle regulator (6, 34). Whether PKM2 regulates the cell cycle in endothelial cells has come under contention, however. Gómez-Escudero et al. (1) did not find knockout of PKM2 to inhibit endothelial proliferation in vivo. This differing result was attributed to compensatory expression of PKM1 as the cause of suppressed endothelial proliferation and not PKM2 itself (1, 123). Further investigation is necessary to clarify if the loss of PKM2 or gain of PKM1 is the cause of endothelial proliferation arrest.

Rapid glycolytic flux during endothelial proliferation is paralleled by acidosis in vitro (4, 17). Little is known about how the endothelium maintains intracellular pH homeostasis during proliferation. It is likely that the expression of monocarboxylate transporters, Na⁺/H⁺ exchangers, V-H⁺-ATPases, and other pH regulatory proteins that extrude excess protons from the cell, maintain the optimal intracellular pH for DNA and protein synthesis, enzyme function, and cell cycle progression (71, 78, 108, 124, 125). Whether metabolic and pH regulatory proteins must be spatially organized during proliferation to prevent intracellular acidosis has yet to be determined, but it is clear they must work together to support fast endothelial proliferation.

Endothelial migration and proliferation are inter-related functions necessary for angiogenesis. Two forms of angiogenesis exist, known as intussusceptive and sprouting angiogenesis. Intussusceptive angiogenesis is a unique form of vascular remodeling that is poorly understood and is denoted as the splitting of one vessel into two vessels (126–129) (Fig. 3C). In sprouting angiogenesis, a new blood vessel forms by branching off an existing vessel (129) (Fig. 3C). This process is dependent on endothelial cells becoming two distinct subtypes called tip and stalk cells (130). The tip cell protrudes filopodia and lamellipodia and navigates vessel migration, whereas the stalk cells quickly proliferate to support vessel elongation (130) (Fig. 3C). These two endothelial subtypes utilize aerobic glycolysis in different manners to fulfill their distinct functions. The migrating tip cell compartmentalizes glycolytic enzymes to the filopodia and lamellipodia to fuel migration (5, 13), whereas proliferating stalk cells localize glycolytic enzymes to the cytoplasm and nucleus, supporting proliferation through ATP generation, biomass production, and cell cycle regulation (2, 5–7, 34, 36–39, 104, 113, 114). The significance of aerobic glycolysis in angiogenesis is evident, as endothelial-specific deletion of HK2, PFKFB3, and PKM2 impairs sprouting angiogenesis in vivo (1–3, 5–7, 34–37).

After a pneumonectomy, sprouting and intussusceptive angiogenesis are markedly increased in the remaining lung (131, 132). Sprouting and intussusceptive angiogenesis are essential processes in the formation of new alveoli and are likely responsible for the compensatory lung growth that occurs post-pneumonectomy (131, 132). Interestingly, angiogenesis and lung growth are not uniform throughout the remaining lung but are localized to the cardiac lobe and discrete subpleural regions in mice (131, 132). This heterogeneity in vascular expansion and lung growth was suggested to be from higher mechanical stretching in these regions since cyclic stretching is a known stimulus of angiogenesis (132, 133). The remaining lung also has elevated radiolabeled glucose uptake in the regions that correlate with localized angiogenesis and lung regeneration (134). These studies indicate endothelial glycolysis is an important mediator of angiogenesis and lung regeneration following a pneumonectomy and requires further inquiry.

In conclusion, aerobic glycolysis fuels endothelial migration, proliferation, and angiogenesis to facilitate neovascularization of damaged alveolar regions and likely contributes to lung regeneration after injury, making aerobic glycolysis a key element in pulmonary vascular repair.

Endothelial Metabolism in Postnatal Angiogenesis

Lung growth following a pneumonectomy was noted to be similar to postnatal lung development since both processes involve extensive expansion of the pulmonary vasculature and new alveoli formation (132). From infancy to adulthood, there is a 20-fold increase in the alveolar-capillary surface, resulting in 480 million alveoli (135) with 86% of the alveolar surface area covered with capillaries (15, 136). At birth, alveolar sac expansion, elevated oxygen tension, vascular endothelial growth factor (VEGF), and nitric oxide (NO) stimulate rapid postnatal angiogenesis in the lung (137–140). PMVECs rely on aerobic glycolysis to meet the bioenergetic demand of angiogenesis in vitro (4, 8) and glycolysis is necessary for retinal postnatal angiogenesis in vivo (5–7, 34), providing evidence that aerobic glycolysis may be a critical component in the maturation of the pulmonary circulation.

In preterm infants, disruption of pulmonary postnatal angiogenesis impairs alveolarization and is suggested to be a contributing factor in bronchopulmonary dysplasia (137, 138, 141). Metabolomic analysis of lung tissue from a bronchopulmonary dysplasia and pulmonary hypertension mouse model shows a reduction in glycolytic metabolites, indicating suppressed glycolysis in the lung (142). Lung endothelial FAO is also knocked down in mice with bronchopulmonary dysplasia, and genetic inhibition of endothelial FAO exacerbates alveolar and vascular growth arrest during hyperoxia (143). The protective mechanism of endothelial FAO against hyperoxia-induced oxidative stress in premature infants is not known but could be related to FAO’s role in angiogenesis and antioxidant defense (10, 88). Thus, endothelial glycolysis and FAO likely have important functions in lung development and bronchopulmonary dysplasia, but their exact role requires more rigorous characterization.

Metabolites in Endothelial Epigenetic Regulation and Cell Signaling

Acetylation and methylation are key post-translational modifications which modulate transcription factor activity and chromatin structure, controlling gene expression (94, 95). The presence or absence of these post-translational modifications is mediated by epigenetic regulatory enzymes whose catalytic reactions depend on TCA cycle metabolites (94, 95). In addition, several TCA cycle metabolites act as enzyme inhibitors (94, 95). Thus, the bioavailability of metabolite cosubstrates and inhibitors control the activity of epigenetic regulatory enzymes and have a major role in dictating the cell’s epigenetic landscape (94, 95) (Fig. 3D).

Acetyl-CoA is a required cosubstrate for acetyltransferase and is supplied through the TCA cycle, making the rate of metabolism a regulator of acetylation (94, 95). The counterparts to acetyltransferases are deacetylases, many of which rely of NAD⁺. Sirtuin 1 (SIRT1) is a prominent NAD-dependent deacetylase in the endothelium and suppresses the antiangiogenic transcription factor FOXO1, mediating angiogenesis after exercise and ischemic injury in mice (144). Interestingly, dietary supplementation of NAD⁺ precursors has garnered attraction as a mechanism to increase the bioavailability of NAD⁺, stimulating endothelial SIRT1 and increasing capillary density in the elderly (144).

The TCA cycle metabolite α-ketoglutarate (α-KG) is a cosubstrate for α-KG dependent dioxygenases, a class of enzymes that catalyze demethylation and hydroxylation reactions (94, 95). During the reaction, α-KG is converted to succinate which then acts as a negative feedback inhibitor (94, 95, 145). Fumarate is another metabolite inhibitor of α-KG-dependent dioxygenase enzymes and accumulation of intracellular succinate/fumarate suppresses these demethylases, causing DNA hypermethylation and altering the cell’s epigenetic landscape (94, 95, 145, 146) (Fig. 3D).

Prolyl-hydroxylases (PHDs) are α-KG-dependent dioxygenase enzymes responsible for the hydroxylation and degradation of hypoxia-inducible factor (HIF)-1α (94, 147). Accumulation of intracellular succinate inhibits PHD activity, preventing HIF-1α hydroxylation and promoting HIF-1 transcription (94, 147). Aortic endothelial cells treated with a membrane permeable form of succinate activates HIF-1 transcription and VEGF production, enhancing angiogenesis and cell migration (148). In line with this, impairing succinate production reduced angiogenesis in vivo (148).

Although succinate is typically associated as an intracellular metabolite, it is also found in plasma where it functions as an extracellular ligand to succinate receptor 1 (SUCNR1) (formerly known as GPR91) (149, 150). Aortic endothelial cells and HUVECs express SUCNR1 and succinate activation of SUCNR1 stimulates VEGF production, augmenting angiogenesis (148). Therefore, both intracellular and extracellular succinate have signaling functions which promote angiogenesis and endothelial migration (148) (Fig. 3D).

Our understanding of intracellular and extracellular metabolite signaling has advanced exponentially over the past decade, but how these principles integrate into pulmonary physiology and disease is unknown. Discovering how the metabolite signature at the alveolar-capillary niche regulates pulmonary endothelial epigenetics and how extracellular metabolites relay signals to these cells may link metabolic health to respiratory function.

SINGLE-CELL RNA-SEQUENCING REVEALS SPECIALIZED LUNG CAPILLARY ENDOTHELIAL SUBPOPULATIONS

Not all endothelial cells contribute evenly to repair, as endothelial cells from pulmonary vessels possess different proliferative potentials (64). To better understand the in vivo relevance of endothelial heterogeneity in vascular function and repair, several research groups utilized single-cell RNA-sequencing on mouse and human lungs to categorize endothelial cells into functional groups. Gillich et al. (151) identified two functional PMVEC populations, termed aerocytes and general capillary endothelial cells, which was confirmed by Schupp et al. (152) (Table 2). Aerocytes rarely proliferate and have four to five times greater surface area than general capillary endothelial cells. They are exclusively positioned at thin regions of the alveolar wall in close proximity to alveolar type I (AT1) cells, suggesting aerocytes are specialized for gas exchange (151). General capillary endothelial cells are located in thick(er) regions of the alveolar wall, indicating they contribute less to gas exchange (151). Instead, 5-ethynyl-2′-deoxyuridine (EdU) incorporation revealed general capillaries are proliferative and may be the progenitor cells that are responsible for repairing the endothelial barrier post injury (151).

**Table 2. Single-cell RNA-sequencing identifies genetically distinct microvascular endothelial cell subpopulations in mouse lung tissue**
General Capillary Cells	Aerocyte	Car4-High Cells
Gpihbp1	Car4	Car4
Plvap	Ednrb	Ednrb
Cd93	Fibin	Fibin
Ptprb	Tbx2	Tbx2
Cemip2	Rprml	Rprml
Tek	Chst1	Chst1
Cxcl12	Apln	Apln
Aplnr	Cdkn2b	lgfbp7
		Emp2
		AW112010
		Pmp22
		Ptp4a3
		Ccdc184
		Clu
		Ccdc68
		Tmeff2
		Cd34
		Enho
		Sept4
		Kd4

RNA analysis reveals three primary mouse lung microvascular endothelial cell subpopulations termed general capillary cells, aerocyte, and Car4-high cells (151, 153). The table lists the top differentially expressed genes in each subpopulation. Notably, aerocyte and Car4-high endothelial cells have similar gene expression. Aerocytes are suggested to be specialized for gas exchange while general capillaries and Car4-high endothelial cells are expected to act as progenitor cells (151, 153). [Adapted by permission from Springer Nature Customer Service Center GmbH: Springer Nature, Nature, Gillich et al. (151) and from Niethamer et al. (153).].

Niethamer et al. (153) also performed single-cell RNA-sequencing and EdU assays on mouse lungs. Their work identified a new endothelial population termed Car4-high cells, due to the high expression of carbonic anhydrase IV (CA IV) (153). This unique endothelial type was determined to be a progenitor cell population that repairs damaged alveolar regions after injury and is primed to receive signals from AT1 cells (153). Interestingly, Gillich et al. (151) grouped cells with high Car4 expression as aerocytes, which are also primed for AT1 cell communication but possess limited repair capacity (Table 2). Further investigation is necessary to clarify if aerocytes and Car4-high cells are the same or different endothelial subpopulations. It also leaves us to consider the function of CA IV, since numerous studies show it is abundantly expressed on the luminal side of pulmonary capillaries (151, 153–155). CA IV was suggested to regulate local pH and carbon dioxide exchange at the alveolar-capillary membrane (154), but it could have other functions that are independent of its catalytic activity. In vitro, CA IX regulates PMVEC wound healing and metabolism in addition to its role in pH homeostasis (71, 80). Whether CA IV has similar noncanonical functions involved in repair has yet to be seen. Nevertheless, CA IV seems to be an important molecule as CA IV knockout mice produced smaller litters than predicted (156).

Moving forward, it will be critical to understand the metabolic signatures of each pulmonary endothelial subpopulation. RNA analysis suggests general capillary endothelial cells and aerocytes have differing roles in lipid metabolism and must coordinate to breakdown lipoprotein triglycerides into fatty acids (151). Reparative cell populations, such as the general capillary and Car4-high endothelial cells, will likely possess metabolic pathways that are tuned for proliferation, positioning glycolytic enzymes in the perinuclear region. On the other hand, aerocytes were suggested to be primed for leukocyte interaction and will likely position glycolytic enzymes at AJs to fuel endothelial membrane remodeling during leukocyte transcytosis. Discovering how these endothelial subpopulations tailor their metabolism to fulfill their specific functions will bring mechanistic insights into the pulmonary endothelium’s heterogenous response to injury and may identify distinct metabolic signatures as novel therapeutic targets to minimize injuries and facilitate repair.

ENDOTHELIAL CELL METABOLISM IN ACUTE RESPIRATORY DISTRESS SYDNROME

Aerobic glycolysis is increased during infection, leading to elevated lactate in the blood (157–163). High serum lactate is a predictor of death in patients with acute respiratory distress syndrome (ARDS) and sepsis (160, 163), and pulmonary lactate production directly correlates with the severity of lung injury (159, 161). Wang et al. (158) examined whether this increase in glycolysis during ARDS is mediated through glycolytic activator PFKFB3. Mice injected with LPS exhibited significant lactate production and elevated PFKFB3 expression in the lung, particularly in CD31-positive endothelial cells (158). Endothelial-specific PFKFB3 knockout mice have a substantial reduction in pulmonary edema, lower cardiac and kidney injury, and an improved survival rate compared with wild-type mice (158). A similar protective effect is seen in wild-type mice treated with the PFKFB3 inhibitor 3PO during LPS-induced injury (158). Mechanistically, endothelial PFKFB3 expression activates NF-κB, inciting leukocyte infiltration and exacerbating endothelial permeability (2, 158, 164). In addition, wild-type mice have fewer VE-cadherins at AJs of lung endothelial cells compared with endothelial-specific PFKFB3 knockout mice (158), suggesting that glycolysis fuels VE-cadherin internalization and destabilizes AJs during LPS treatment.

Glycolysis, however, is not necessarily detrimental in all scenarios of vascular injury. Following endothelial permeabilization, Cheung et al. (165) found the endothelial CD31 receptor activates Akt, inducing a robust glycolytic response that sustains the energy required for actin remodeling and re-annealment of AJs. CD31-deficient mice had excessive vascular leakage in the lungs and other organs compared with wild-type mice 6 h postinjection of a vascular permeabilizing agent (165). Treatment with an Akt activator rescued the robust glycolytic response in endothelial cells and corrected vascular leakage in CD31-deficient mice (165). Thus, inhibiting glycolysis may defend against pulmonary vascular injury by lowering the inflammatory response and stabilizing AJs during the immediate injury (2, 158, 164), but glycolysis is necessary to facilitate repair and barrier restoration during the recovery phase (165). These studies, while informative, leave several gaps in our knowledge. Inhibiting glycolysis is clearly beneficial during LPS injury, but whether this is true during live microbial infections is not known and requires further investigation. In addition, optimizing the potency of glycolytic inhibitors, identifying biomarkers to guide the timing of glycolytic inhibition or enhancement, and understanding the heterogenous metabolic response of pulmonary endothelial subpopulations are fundamental questions that need to be addressed to pursue endothelial metabolism as a novel therapeutic avenue in ARDS and sepsis.

ENDOTHELIAL METABOLISM IN CORONAVIRUS DISEASE (COVID-19)

With the emergence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the cases of ARDS have surged worldwide (166). Consequently, endothelial dysfunction is garnering more appreciation as a major contributor to ARDS and multiorgan failure following COVID-19 (126, 167, 168). SARS-CoV-2 incites vascular inflammation (endotheliitis), permeabilizes the endothelial barrier, facilitates leukocyte infiltration, and promotes hypercoagulation (168). Teuwen et al. (168) proposed the manipulation of endothelial metabolism as one possible therapeutic route in COVID-19 associated ARDS. Blocking PFKFB3 prevents an exaggerated glycolytic response in endothelial cells, preventing VE-cadherin internalization and suppressing NF-κB signaling (2, 5, 158, 164). Together, these effects identify PFKFB3 as a possible target to reverse endotheliitis, improve vascular integrity, and reduce leukocyte infiltration into the alveolar space in COVID-19 associated ARDS.

COVID-19 pneumonia lungs are histologically distinguishable from those of influenza pneumonia due to several unique vascular features, including a ninefold increase in pulmonary capillary microthrombi and intussusceptive angiogenesis (167). The close association of microthrombi and microvessel remodeling has led several others to hypothesize that intussusceptive angiogenesis acts as an adaptive mechanism that navigates blood flow around clotted vessels (169). How endothelial cells meet the metabolic demand of intussusceptive angiogenesis is unknown. However, endothelial cells, in general, rely on glycolysis to drive migration and vascular remodeling (1–3, 5–7, 34–40) and are likely the primary bioenergetic pathway. Studies on the metabolic requirements of intussusceptive angiogenesis and its concomitant function in infection will aid our understanding in how metabolism drives pulmonary vascular remodeling in COVID-19 and other infections.

Aberrant remodeling of the pulmonary circulation can cause pulmonary hypertension (PH), which is a highly prevalent complication in patients with ARDS with studies reporting PH in 46% to 92% of ARDS cases (170–172). Clinically, PH is defined as a mean pulmonary arterial pressure greater than 20 mmHg and can occur in patients with ARDS due to lung injury and hypoxia (173). In hospitalized patients with COVID-19, PH was present in 12% of cases and was associated with a worse outcome compared with patients with COVID-19 without PH (174). Histological assessment of deceased patients with COVID-19 revealed thickening of the pulmonary vascular walls, a feature commonly seen in pulmonary arterial hypertension (PAH) (175). Another study evaluated the presence of PH and right ventricular dysfunction in young patients recovering from moderate COVID-19 infections 2 mo after hospital discharge (176). Each patient was under the age of 55 with no history of cardiovascular disease (176). In this report, PH was prevalent in 7.7% and right ventricular dysfunction was present in 10.3% of patients (176). Together, these studies suggest patients with COVID-19 are at risk for developing PH during the acute infection and as a long-term complication.

Several mechanisms can cause PH in ARDS, including thrombosis. Since microthrombi are unique histological feature of COVID-19, it is possible thrombosis is a primary cause of PH in patients with COVID-19. It is important to note, however, that patients with COVID-19 on anticoagulants (176) still developed PH, indicating other factors such as microvascular injury and vasomotor dysregulation may contribute to COVID-19 PH. The renin-angiotensin-aldosterone system (RAAS) is an important regulator of blood pressure and activation of RAAS leads to the release of angiotensin II, a potent vasoconstrictor and mediator of inflammation (177). Angiotensin II is broken down via the angiotensin converting enzyme 2 (ACE2) which acts as a negative regulator to RAAS (177, 178). Paradoxically, since ACE2 is also a SARS-CoV-2 receptor, it has been speculated whether chronic downregulation of ACE2 in PAH lungs (179) protects the PAH population against COVID-19 (180, 181). Many caution not over interpret this, however, suggesting more clinical data are required to accurately evaluate the PAH population’s risk for severe COVID-19 infection (182).

During SARS-COV-2 infection, ACE2 is downregulated, potentially lowering the system’s brake and is suggested to contribute to COVID-19 pathology (178, 183). Nonsurviving patients with COVID-19 (184–186) and sepsis (187–190) present with metabolomic abnormalities, including elevated plasma succinate. Interestingly, high plasma succinate stimulates SUNCR1 in kidney cells, activating RAAS (150). Whether SUNCR1 activation and ACE2 downregulation lead to RAAS dysfunction in COVID-19 is not known and further work is necessary to determine their role in COVID-19-induced PH.

Pulmonary vascular injury during the acute phase of COVID-19 can lead to pulmonary artery remodeling, further exacerbating the hypertensive effects with potential long-term consequences (177). Recently, glycolysis was determined to be a mediator of progressive pulmonary vascular remodeling (191). PAECs from rodents with PH and patients with idiopathic PAH upregulate PFKFB3 to increase the rate of glycolysis (191). Knockout and pharmacological inhibition of PKFBF3 protects mice from developing PH under 5 wk of hypoxia and during hypoxia/sugen treatment (191). Mean pulmonary arterial pressure and right ventricular systolic function are dramatically improved while pulmonary artery wall thickening and right ventricular remodeling are nearly abolished in endothelial-specific PFKFB3 knockout mice (191). The mechanistic reason for protection is multifactorial. Upregulation of PFKFB3 increases the rate of glycolysis, producing several metabolites that stabilize HIF2a expression (191). HIF2a then promotes PAEC production of inflammatory molecules and growth factors, causing lung inflammation and pulmonary artery smooth muscle proliferation (191). Higher glycolytic rates also generate larger quantities of ATP to support vessel remodeling (5) and preventing PFKFB3 activity ameliorates these effects. Thus, targeting PFKFB3 may prove as effective strategies to reduce acute and long-term PH in patients with COVID-19.

CONCLUSIONS

The pioneering work of Pasteur, Meyerhof, and Warburg during the eighteen- and nineteen-hundreds laid the groundwork necessary to discern how aerobic glycolysis supports endothelial cell function. Our understanding of the endothelium has come far since the 1960s, progressing from an inert barrier to a highly metabolic tissue. Here, we discussed how endothelial metabolism preserves pulmonary vascular homeostasis by 1) regulating endothelial permeability, 2) providing antioxidant defense, 3) facilitating pulmonary vascular repair, 4) driving postnatal angiogenesis, and 5) producing metabolites for epigenetic regulation and cell signaling. These emerging functions have significant physiological and clinical implications pertaining to ARDS, sepsis, and COVID-19. In particular, targeting glycolysis during infection stabilizes the pulmonary vascular barrier, dampens inflammation and leukocyte infiltration, and facilitates repair (1, 6, 158, 165). Several lapses in our knowledge still remain, however. The dosage and timing of glycolytic inhibition during lung infection requires thorough study to efficiently target aerobic glycolysis in ARDS. In addition, characterizing the distinct metabolic profiles of each pulmonary endothelial cell subpopulation will bring insight into the heterogeneity of lung injuries and may identify targetable metabolic systems in reparative cells. The metabolomic profile of many critically ill patients is indicative of metabolic abnormalities (184–189) and surviving patients with ARDS experience long-term lung injury (192). Whether patients with ARDS have lasting metabolic dysfunction in endothelial cells, inciting long-term maladies is another question that remains to be investigated. Addressing these questions and continuing fundamental studies in pulmonary endometabolism will bring forward new directions for improving the short- and long-term health of patients with ARDS and other pulmonary diseases.

GRANTS

This work was supported in part by American Heart Association Grant 18CDA34080151 (to J. Y. Lee) and National Heart, Lung, and Blood Institute Grants HL-66299, HL-60024, and HL-148069 (to T. Stevens).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.P.S. prepared figures; R.P.S., S.S.P., and S.C.J. drafted manuscript; R.P.S., T.S., and J.Y.L. edited and revised manuscript; R.P.S., T.S., and J.L. approved final version of manuscript.

ACKNOWLEDGMENTS

The authors thank Drs. Mike T. Lin, Wito Richter, and Ron Balczon for helpful critiques of the manuscript.

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AUTHOR NOTES

Correspondence: J. Y. Lee (jlee@health.southalabama.edu).

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American Journal of Physiology-Lung Cellular and Molecular Physiology 321 2 cover image

Volume 321Issue 2August 2021Pages L358-L376

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History

Received 29 March 2021

Accepted 15 June 2021

Published online 10 August 2021

Published in print 1 August 2021

Keywords

Endothelial metabolism in pulmonary vascular homeostasis and acute respiratory distress syndrome

Abstract

INTRODUCTION

THE DISCOVERY OF AEROBIC GLYCOLYSIS AS A MECHANISM TO SUPPORT ATP SYNTHESIS IN PULMONARY MICROVASCULAR ENDOTHELIAL CELLS

The History of Aerobic Glycolysis

Aerobic Glycolysis in the Pulmonary Endothelium

ENDOTHELIAL METABOLISM PRESERVES PULMONARY VASCULAR HOMEOSTASIS

Aerobic Glycolysis Regulates Pulmonary Vascular Permeability

Endothelial Fatty Acid Oxidation as an Antioxidant Defense

Endothelial Glycolysis Facilitates Pulmonary Vascular Repair and Lung Regeneration

Endothelial Metabolism in Postnatal Angiogenesis

Metabolites in Endothelial Epigenetic Regulation and Cell Signaling

SINGLE-CELL RNA-SEQUENCING REVEALS SPECIALIZED LUNG CAPILLARY ENDOTHELIAL SUBPOPULATIONS

ENDOTHELIAL CELL METABOLISM IN ACUTE RESPIRATORY DISTRESS SYDNROME

ENDOTHELIAL METABOLISM IN CORONAVIRUS DISEASE (COVID-19)

CONCLUSIONS

GRANTS

DISCLOSURES

AUTHOR CONTRIBUTIONS

ACKNOWLEDGMENTS

REFERENCES

AUTHOR NOTES

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