Chapter 3　Mechanisms of Hepatopulmonary Syndrome

Hepatopulmonary syndrome (HPS) is a progressive disease characterized by worsening hypoxemia due to intrapulmonary vascular dilatation (IPVD), arteriovenous malformations and increased vasoactive substances in the setting of chronic liver disease (CLD) ^{1, 2}. Prevalence of HPS varies from 4%～47% due to different cut-offs in defining arterial hypoxemia in primary studies, and mortality rate of HPS is about 41% ³. Over the past two decades, the pathogenesis and precise mechanisms of HPS were under active investigation. Based on the experimental and clinical research, the mechanisms of HPS continue to be uncovered, which thus provides the chance of clearly understanding the HPS pathogenesis and potential therapeutic targets. Although progress has been made in delineating the mechanisms underlying the imbalance of vasoactive substances, pulmonary vascular alterations and angiogenesis in HPS, to date, there is still lack of related effective therapeutic approaches apart from liver transplantation (LT) ^{4, 5}. In this chapter,we will review and summarize current concepts regarding the mechanisms of HPS to provide a more comprehensive understanding of the HPS pathogenesis (Figure 5).

It has been reported that the pathogenesis of pulmonary microvascular changes includes the alterations in liver that affect lung and the direct changes in lung, such as pulmonary vasodilation, intravascular monocyte accumulation and angiogenesis ^6-8. Previous studies have identifed a sequence of molecular alterations in the pulmonary vasculature that infuence vascular tone during the onset and progression of HPS ^9-12. Lung, an essential organ downstream of liver, functions as the most important site for the metabolism of various vasoactive substances.Here, we will list four different vasoactive substances and briefly clarify their concrete mechanisms.

Nitric oxide, a powerful pulmonary vasodilator linked to vascular abnormalities, has been identifed as a crucial mediator of impaired oxygenation in patients with HPS ^{13, 14}. NO, an inorganic free radical gas, is endogenously biosynthesized from L-arginine and oxygen by two different nitric synthase (NOS) enzymes, that are inducible NOS (iNOS) and endothelial NOS(eNOS), both of which are highly expressed in the pulmonary capillaries ¹⁵. Overproduction of pulmonary NO is attributed to the increased levels and activities mainly of iNOS, to a lesser extent of eNOS, within pulmonary intravascular macrophages. Due to the intestinal bacterial translocation and the weakened detoxifcation function of liver, the augmented endotoxins and cytokines in circulation, such as TNF-α, lead to an elevated level of NO through the activation of iNOS. In addition, NO overexpression is partly attributed to the enhanced pulmonary eNOS synthesis, which is activated through the receptor-dependent endothelin-1 (ET-1) ¹⁶. It is worth noting that the increased vascular shear stress, which is induced by hyperdynamic circulation in cirrhosis patients, also can upregulate eNOS expression. Results of both experimental and clinical research indicate that NO overproduction results in both pulmonary and systemic vasodilatation,which further confrms the close relationship between NO and intrapulmonary vascular dilatation in patients with HPS. Based on a before-and-after observational study, Rolla G et.al found that the exhaled NO concentrations are increased accompanied with a widened alveolar-arterial oxygen gradient in HPS patients, while NO concentrations return normal after orthotropic liver transplantation ¹⁷. Chronic pharmacological inhibition of pulmonary iNOS- and eNOS-derived NO production with methylene blue, inhibition of the NO activity through soluble guanylate cyclase or N ^G-nitro-L-arginine methyl ester (L-NAME), and inhibitors of NOS, transiently improve hypoxemia in HPS patients ^{18, 19}. A large number of studies suggest an essential role of endothelial NO for postnatal neovascularization, which is consistent with the observation that the eNOS-knockout mice are characterized by an impaired angiogenesis after administration of VEGF ^20-22.In addition to the important role of NO for IPVD and angiogenesis, NO has been shown to exert both proliferative and anti-proliferative effects on pulmonary vascular cells ²³. It is worth noting that the origin site and concentration of NO may affect the role of NO. Previous studies have demonstrated that exogenous NO inhibits pulmonary vascular remodeling, while endogenous NO derived from eNOS and iNOS functions as an important mediator of pulmonary vascular angiogenesis. Taken together, all of these evidences suggest that endogenous NO is functionally important during the progression of HPS.

ET-1, one of three isoforms of endothelin, is mainly produced by the activated endothelial cells (ECs) and identifed as a well-recognized vasoconstrictor ²⁴. ET-1 serves as a local paracrine and autocrine regulator of vascular tone both under physiological conditions and in patients with cirrhosis, and its function is mediated by two G protein coupled receptors (ET _A and ET _B).Endothelial A receptor (ET _A) is mainly located in the vascular smooth muscle cells (VSMCs),and it mediates vasoconstriction. Endothelial B receptor (ET _B), the dominant subtype of ET-1 receptors, is classified into two different types: one in ECs that stimulates the expression of eNOS and NO, and the other in SMCs that mediates vasoconstriction ²⁵. A working model has established to study the potential effects of ET-1 on the pulmonary microcirculation. In experimental HPS, bile duct stellate cells as well as proliferating cholangiocytes function as the major source of ET-1 production, and the production of ET-1 in cholangiocytes might be dependent on the transforming growth factor β (TGF-β)-mediated signaling, based on the p-Smad2 localization and levels accompanied with ET-1 ^{26, 27}. Then, excessive ET-1 moves into blood circulation to stimulate lung microvascular eNOS activation as well as monocyte accumulation through binding with ET _B receptor that is selectively increased in pulmonary vascular endothelial cells, fnally leading to pulmonary vasculardilation and the development of a widened alveolar-arterial oxygen gradient ²⁸. Based on the experimental and clinical research, emerging evidence suggests that excess of ET-1 production in the plasma, liver and lung tissue is closely associated with intrapulmonary vasodilatation in HPS, which is mainly attributed to its non-vasoconstrictor function including an enhanced eNOS synthesis through a calcium-mediated pathway independent of Akt activation or an increased expression of vasodilatory peptides, including tumor necrosis factor-α (TNF-α), vasoactive intestinal peptide(VIP) and prostaglandin (PG) ¹⁶. In addition, the increased TNF-α can directly modulate monocyte adhesion and infuence ET _B receptor expression as well as NO production in endothelial cells,which suggests that ET-1 and TNF-α interact with each other in the development of HPS ²⁹.ET _B receptor has been identifed as the dominant subtype of the ET-1 receptors in pulmonary endothelial cells, and the selective defcient of ET _B receptor reduces lung Akt/eNOS activation,and thus ameliorates experimental HPS ³⁰. In summary, these observations establish an important role of ET-1/ET _B signaling in the onset and progression of HPS.

Carbon monoxide, a potent vasodilator, is enzymatically synthesized by heme oxygenase-1(HO-1). CO shares many characteristics with NO, and it also participates in the pathogenesis of HPS ^{11, 31}. HO-1 serves as the primary source of the circulating CO and contributes to vasodilation through the HO-1 derived CO ³². Activity of HO-1 can be induced by various stimuli,including hypoxia, endotoxin as well as ischemic-reperfusion injury. Accumulating evidence from experimental and clinical studies has demonstrated that expression levels of HO-1 in pulmonary vascular macrophages are elevated concurrently with the increased CO production as HPS progresses. Carter et al. has demonstrated that overproduction of NO upregulates the expression levels of HO-1 in the lung of CBDL rats via a cyclic GMP-independent mechanism,and the CO derived from excessive HO-1 finally leads to the pulmonary vascular dilatation ¹¹.After administration of ZnPP-IX, a specifc HO-1 inhibiting enzyme, pulmonary vasodilation is attenuated as well as symptom of HPS is improved. In addition, after administration of L-NAME,an inhibitor of NO, both expression levels of HO-1 and vascular vasoconstriction are normalized.These findings suggest that endogenous NOS/NO and HO-1/CO systems contribute to the development of HPS, either independently or synergistically.

Estradiol and progesterone, two well-known vasoactive substances, are significantly elevated in cirrhotic patients and are prominently effective in the pulmonary vasculature ¹². In human patients, elevated levels of both estrogen and progesterone are observed in hypoxemia and pulmonary vascular dilatation. In addition, chronic liver diseases often have an imbalance of serum estradiol and progesterone. The higher levels of serum estradiol are mainly due to the increased peripheral aromatization of testosterone to estrogens because of the impaired liver metabolism. The higher levels of serum progesterone are also attributed to the impaired liver metabolism; in addition, the increased biological effects also can come from the increased progesterone receptor secondary to estradiol. After liver transplantation, sex hormones levels are back to normal, which indicates that estradiol and progesterone may play an important role in the pathogenesis of HPS ³³.

Estradiol can dilate blood vessels in both reproductive and non-reproductive tissues due to the activation of iNOS thus leading to an excessive production of the potent vasodilator NO or blocking calcium-channel. Interestingly and consistently, L-NAME, an inhibitor of NOS,could abrogate estrogen vasodilation ^{34, 35}. Increased estrogen levels cause the spider naevi, which has been observed in pregnant females as well as cirrhotic patients. After administration of estrogen, both vascular dilatation and increased NO levels are observed in rats. Conversely, in the oophorectomized rats, pulmonary vascular dilatation and hypoxemia are improved concurrently with a decrease in estrogen and NO levels ³⁶. Serdar Yol et al. further demonstrated that serum estrogen levels have a positive correlation with serum NO levels as well as perialveolar vessel diameters, but a reverse correlation with O ₂ saturation levels ³⁴. Progesterone can lead to a dosedependent but endothelium-independent relaxation of vessels, which is mediated by a receptoractivated 3', 5'-cAMP mechanism ¹². In summary, the higher levels of estradiol and progesterone contribute to the pathogenesis of HPS, especially pulmonary vasodilatation.

More and more evidence from experimental and clinical research indicates that intestinal endotoxemia, with a high incidence in a variety of diseases, is mainly attributed to intestinal barrier dysfunction and increased gut permeability; and intestinal endotoxemia represents an important common pathogenic factor in the progression of various complications of cirrhosis,such as HPS and hepatorenal syndrome ^{37, 38}. In physiology conditions, pulmonary vascular bed isn't easily exposed to bacterial products, which are normally filtered out by liver phagocyte.In a variety of pathological situations, lung functions as an important site to clean bacteria and endotoxins and it is sensitive to intestinal endotoxemia, thereby easily leading to the various distant-focus infections ³⁹. In cirrhosis, the decreased capacity of liver phagocyte results in the increased levels of circulating bacterial endotoxins; then the elevated bacterial endotoxins enter the pulmonary circulation, and fnally are complementally cleaned up by increased pulmonary phagocytic activity ⁴⁰. The increased activity of pulmonary phagocyte is attributed to extensively accumulated pulmonary intravascular macrophages, which mainly adhere to pulmonary endothelium. Under the stimulation of endotoxins, the activated pulmonary intravascular macrophages release various infammatory cytokines and vasoactive substances, such as TNF-α,NO, ET-1 and HO-1 ³⁹.

TNF-α, a pleiotropic cytokine, is involved in various diseases, including inflammation,cancer, immune response and so on. As mentioned above, levels of TNF-α are increased in cirrhosis patients induced by intestinal endotoxemia. Inhibition of TNF-α could inhibit iNOS and LPS-induced pulmonary infammation through TNF-α-PI3K/Akt-NO signaling pathway. Liu et al. demonstrated that a specifc monoclonal antibody to TNF-α (TNF-α McAb) could decrease circulating endotoxin concentration and NO production, mitigate lung inflammation, reduce macrophage adherence, and fnally improve intrapulmonary shunt in experimental HPS. Taken together, these indicate that endotoxin-TNF-α- PI3K/Akt-NO signaling pathway is activated in HPS ²⁹.

The plasma levels of LPS, a major molecule in the outer membrane of Gram-negative bacteria, are markedly elevated in patients with HPS and are highly correlated with pulmonary infammation, intrapulmonary vasodilation and worsening hypoxemia, which further suggests that IETM and its associated infammatory cytokines (IL-1, IL-6, TNF-α) contribute to pathogenesis of HPS ³⁷. Administration of norfloxacin, a quinolone predominantly active against Gramnegative bacteria, prevents bacterial translocation, inhibits endotoxin injury to the lung, reduces the migration of macrophages to lung circulation, decreases the activity of NOS, and improves the symptoms of HPS ⁴¹. Moreover, Eduardo Gaio et.al further demonstrated that administration of levofloxacin, another prophylactic antibiotic against gram-negative bacteria, can improve pulmonary vascular remodeling and also attenuate the chest wall and lung heterogeneities as HPS develops ⁴². In 2011, Stelios F. Assimakopoulos et.al proposed a totally novel perspective about IETM that intestinal oxidative stress appears to be critically important for gut barrier dysfunction in cirrhosis-related complications through several aspects, such as enhanced enterocyte apoptosis,modified intestinal tight junctions and impaired intestinal brush border function; and thus they drawed a conclusion that antioxidants could be the “healing elixir” for cirrhosis-related complications, including hepatopulmonary syndrome ⁴³.

It is important to evaluate whether and how intravascular macrophages accumulate and mediate adhesion in the pulmonary vasculature as HPS develops ¹. In the above part we have mentioned that in the pathogenesis of HPS, augmented accumulation of pulmonary intravascular macrophages (PIMs), which is mainly caused by the observed IETM, increases circulating TNF-α,as well as specific chemokine/chemokine receptor pairs such as monocyte chemoattractant protein-1 (MCP-1)/CCL2-CCR2, macrophage inflammatory protein-1 alpha (MIP-1α)/CCL3-CCR1 and fractalkine/CX3CL1-CX3CR1, adheres in the pulmonary microvessel endothelium to stimulate the activity of pulmonary phagocytes, and contributes to a shift of phagocytosis from liver to lung in order to clean up bacteria and toxins in blood ⁴⁴. These PIMs derived from circulating monocytes secrete a variety of vasodilatory, angiogenic as well as proliferative growth factors, including NO, CO, VEGF, TNF-α, platelet-derived growth factor (PDGF) and so on in pathological conditions, such as atherosclerosis, tumor vasculargenesis and some other pulmonary vascular diseases. Previous studies have emphasized the role of accumulated PIMs in the pathogenesis of HPS: overproduction of pulmonary NO refects the increased iNOS levels in PIMs; inhibition of HO-1 reduces the elevated CO production by PIMs; pulmonary angiogenesis suggests the activation of VEGF-mediated signaling pathways in PIMs ⁴⁵. Thenappan and colleagues reported that depletion of these activated PIMs improves the typical symptoms of experimental HPS, such as the widened alveolar-arterial oxygen gradient, abnormal capillary dilatation and increased capillary density ⁴⁶. In addition, depletion of PIMs reduces the activity of extracellular signal regulated kinase (ERK), which is identifed as an important mediator in the angiogenesis signaling pathways.

Fallon and colleagues found that the increased circulating as well as pulmonary chemokine fractalkine (CX3CL1) directly mediates PIMs adhesion, activates VEGF-A production and promotes angiogenesis via CX3CR1 receptor during infammatory angiogenesis after CBDL ⁴⁴.A variety of factors, such as TNF-α, vascular shear stress and ET-1/ET _B receptor activation, can infuence the expression levels of CX3CL1 and CX3CR1. In 2014, Fallon et. al demonstrated that the ET-1/ET _B receptor activation induces CX3CL1 expression through Ca ²⁺ and MEK/ERK-mediated intracellular signaling pathways, finally leading to PIMs accumulation, pulmonary vasodilation and angiogenesis ⁴⁷(Figure 6). In addition, CX3CL1/CX3CR1 signaling has been demonstrated to contribute to experimental HPS through directly activating its related proangiogenic pathways, including VEGFA/VEGFR-2, Raf/MEK/ERK as well as PI3K/Akt/eNOS,in the pulmonary endothelium. Several lines of evidence suggest that pentoxifylline (PTX),a phosphodiesterase and nonspecific TNF-α inhibitor, inhibits PIMs accumulation, reduces pulmonary angiogenic factors and ameliorates the symptoms of HPS, which further emphasizes a positive feedback loop between infammatory cytokines (especially TNF-α) and PIMs during the development of HPS ^{48, 49}. Above all, these fndings emphasize the important roles of PIMs as well as its related chemokine alterations in stimulating pulmonary phagocytic activity and pathological angiogenesis as HPS develops.

In recent years, the progressively increased angiogenesis in the pulmonary microvascular beds as well as the increased pulmonary vessels and capillaries have been observed in both CBDL rat model and HPS patients, suggesting vascular structure alterations including vascular remodeling, vasculogenesis and angiogenesis in the pulmonary vascular bed as HPS develops ⁴⁵.Vascular remodeling is a long-term adaptive response of the vessel wall in response to chronic alterations in blood fow. Vasculogenesis is a process in which endothelial progenitor cells are recruited for the de novo synthesis of vessels ⁵⁰. Angiogenesis, a highly complex and regulated process of new vessels and capillary networks, occurs through the proliferation of endothelial cells (ECs) and smooth muscle cells (SMCs). Angiogenesis is mainly attributed to PIMs accumulation, VEGF-A-dependent signaling pathways in the pulmonary microvasculature, as well as the activation of Akt, ERK and eNOS in the pulmonary endothelium ^{45, 51}. Different from physiological angiogenesis, angiogenesis in the pathological conditions is mainly associated with abnormal capillary networks, whose characteristics include malformation, highly permeability and unstability. The increased pulmonary vessels and capillaries are positively correlated with dynamically increased levels of growth factors, adhesion molecules, chemokine/fractalkine and infammatory cytokines, such as VEGF-A, eNOS, CX3CL1 and TNF-α ^{47, 52}. It has been reported that overproduction of NO in HPS patients induces the increased levels of VEGF, which has been regarded as an important mediator of angiogenesis, monocyte/macrophage accumulation and maintenance of lung barrier function ⁵².

VEGF is a ～23 kDa glycoprotein which includes six members including VEGF-A, VEGF-B,VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PLGF). Among these six members,VEGF-A is the most well characterized one. VEGF-A has different isoforms in humans, including VEGF ₁₂₁, VEGF ₁₄₅, VEGF ₁₆₅, VEGF ₁₈₉, and VEGF ₂₀₆, and VEGF ₁₆₅ is the predominant isoform in many pathological situations ⁵³. Overproduction of VEGF-A occurs in proliferating cholangiocytes and PIMs after CBDL, and the increased expression levels of VEGF-A is also known to induce the cholangiocyte proliferation through an autocrine loop involving ERK activation ⁵². The VEGF angiogenic signaling occurs through its binding with specific receptor tyrosine kinase (RTK)receptors, such as VEGFR-1 and VEGFR-2, distributed on the membrane surface of vascular endothelial cells and other non-endothelial cell types, including monocytes/macrophages, smooth muscle cells and fbroblasts ⁵⁴. It has been reported that activation of VEGFR-2 could trigger its downstream angiogenic signaling pathways, such as Akt and ERK. Sorafenib, a multispecific RTK inhibitor, can improve pulmonary vascular abnormalities and hypoxemia in experimental HPS, by blocking ERK activation and cell proliferation, inhibiting ET-1 production, and reducing eNOS activation and PIMs accumulation in lung, which has identifed a pivotal mechanism in cholangiocytes through which RTK inhibition improves HPS and established RTK-mediated effects on angiogenesis ⁵⁵.

Although some other factors, such as fbroblast growth factor, TGF-β, hepatocyte growth factor and angiopoietins, are also identifed as the important angiogenic regulators, angiogenesis has been thought to be mainly regulated by VEGF.

At the cellular level, our team found that CBDL rat serum induces the proliferation of both PMVECs and PASMCs in vitro, which could contribute to angiogenesis associated with HPS.More specifcally, CBDL rat serum downregulates the expression levels of Annexin A1, a Ca ²⁺-dependent phospholipid binding protein, and thus increases the expression levels of cytoskeleton proteins (such as Destrin, α1-actin and α1-tubulin) within PMVECs, leading to the enhanced cell proliferation. Oppositely, CBDL rat serum upregulates the expression levels of Annexin A2,and thus promotes the phenotypic modulation and proliferation of PASMCs through ERK1/2 and NF-κB signaling pathway ⁵⁶. Based on the analysis of gene expression regulation, we further demonstrated that miR-206 inhibits the HPS rat serum-induced phenotypic modulation and the excessive proliferation of PASMCs by its binding to the 3'-UTR of the ANXA2 mRNA ⁵⁷. In addition, the cross-talk between proliferation and apoptosis of PMVECs has also been identifed as a novel mechanism of angiogenesis. Zhang and colleagues found that the expression levels of 78 kD glucose-regulated protein (this protein is abbreviated as GRP78, and it is a biomarker of endoplasmic reticulum [ER] stress) are elevated in experimental HPS and are positively correlated with those of vascular markers, including FⅦⅧ-RAg and VEGF, suggesting a crucial role of GRP78 in pulmonary microvascular remodeling of HPS ⁵⁸. At the same time, they demonstrated that the increased levels of GRP78 promote pulmonary microvascular remodeling and result in ventilation/perfusion imbalance through promoting cell proliferation and survival through the VEGF-dependent pathway, preventing the caspase-12 release from ER as well as inhibiting apoptosis. Several lines of evidence suggest that the self-repair mechanisms, evoked by initial pulmonary injury, promote the molecular alterations in pulmonary vasculature, such as NO, CO,VEGF-A and the chemokine fractalkine, thus leading to PMVECs proliferation and pathological angiogenesis during the progression of HPS. Based on the important role of initial pulmonary injury in HPS, our team demonstrated that early administration of caspase-3 inhibitor Z-DEVDFMK inhibits the subsequent activation of pulmonary Akt and ERK1, prevents pulmonary angiogenesis and vasodilation, and finally improves arterial oxygenation and alleviates initial lung injury in experimental HPS ⁵⁹. These fndings further emphasize the important role of initial lung injury and its associated molecular alterations in the pathological angiogenesis of HPS.

Recently, the role of gene polymorphisms in regulating the HPS-associated angiogenesis has received much more attention ⁶⁰. In 2010, Roberts and colleagues performed a high-throughput candidate gene study and they identifed some variations associated with the HPS risk in specifc genes, including CAV3, ENG, NOX4, ESR2, VWF, RUNX1, COL18A1 and TIE1 ⁶¹ . However, there is still lack of both suffcient data and further research. Future studies might be focused on the duplication in other populations and the detailed mechanisms in order to elaborate the correlations between single nucleotide polymorphisms (SNPs) of interest and HPS.

WSS induced by sustained blood fow is thought to maintain equilibrium in the pulmonary vasculature. ECs, which are in a closely contact with blood, constantly sense the state of blood flow via WSS, and then transmit the flow information to the SMCs layer in order to maintain baseline WSS values by triggering vasoconstriction or vasodilation ⁶². Chronic alterations to the pulmonary fow can change the vasoregulatory states, fnally leading to the structural remodeling of blood vessels. It has been reported that the elevated shear stress along the ECs wall could keep calcium channels open, which promotes vascular remodeling through complex molecular mechanisms, including various ion channels, growth factor receptors, G proteins, adhesion proteins, glycocalyx as well as cytoskeleton ⁶³. In summary, WSS may play a signifcant role in the HPS-associated vascular remodeling and angiogenesis. However, more extensive research is required to elucidate the concrete mechanism of WSS in the pathogenesis of HPS.

Massive ascites, a major complication of decompensated cirrhosis, markedly increase intra-abdominal pressure (IAP), and finally lead to intra-abdominal hypertension (IAH) ⁶⁴.Recent studies suggested that IAH could cause the impaired liver function, intestinal bacterial translocation, endotoxemia as well as the monocyte/macrophage activation during the development of HPS ^{65, 66}. Zhang and colleagues established a novel IAP mouse model to recapitulate the pathogenesis of HPS. Based on this model, they found that macrophages accumulate in alveolar spaces, alveolar walls become widened, visible blood stagnates in alveolar walls, and a large number of red blood cells extravagate into air space when IAP ranges between 10 and 20 cmH ₂O ⁶⁷. This study suggests that IAH serves as a significant pathological mechanism of HPS. However, its concrete mechanism remains unclear and still requires further evaluation.

Alveolar compartment is consisted of type Ⅰ (AT1) and type Ⅱ (AT2) alveolar epithelial cells, and these two types cells over around 96% and 4% regions of internal air space respectively.AT1 cells form the air-blood barrier for gas exchange and AT2 cells contribute the maintenance of lung function. AT2 cells can produce four different pulmonary surfactants associated proteins(SPs) including SP-A, SP-B, SP-C and SP-D, among which SP-A is the most abundant protein.Pulmonary surfactant can increase lung compliance, reduce surface tension, and inhibit lung collapse ⁶⁸. Both impaired AT2 cell integrity and decreased surfactant protein production are associated with various lung diseases, such as chronic obstructive pulmonary disease (COPD) and acute respiratory distress syndrome (ARDS). Over the past decades, researchers mainly explored the dilation and angiogenesis that occur within pulmonary microvascular compartment. In 2014,Yang and colleagues frstly evaluated the role of alveolar epithelial compartment in experimental HPS ⁶⁹(Figure 7). They observed a selective decrease in the expression levels of pulmonary surfactant protein accompanied by the apoptosis of AT2 cells and a reduction in alveolar airspace,fnally resulting in the ventilation mismatch and abnormal gas exchange as HPS develops after CBDL. In addition, they hypothesized that the increased levels of bile acids or the synergetic effects between bile acids and TNF-α contribute to the selective reduction in AT2 cells during the progression of HPS. Above all, AT2 cell as well as its associated pulmonary surfactant proteins provides a novel focus for the pathophysiology of HPS. However, its concrete mechanism still requires further studies.