Abstract
Hepatopulmonary syndrome (HPS) is a severe complication of cirrhosis with increased risk of mortality. Pulmonary microvascular alterations are key features of HPS; but underlying mechanisms are incompletely understood, and studies on HPS are limited to rats. Placental growth factor (PlGF), a proangiogenic molecule that is selectively involved in pathological angiogenesis, may play an important role in HPS development; however, its role has never been investigated. In this study, we validated an HPS model by common bile duct ligation (CBDL) in mice, investigated the kinetic changes in pulmonary angiogenesis and inflammation during HPS development, and provide evidence for a novel therapeutic strategy by targeting pathological angiogenesis. Mice with CBDL developed hypoxemia and intrapulmonary shunting on a background of liver fibrosis. Pulmonary alterations included increased levels of proangiogenic and inflammatory markers, which was confirmed in serum of human HPS patients. Increased PlGF production in HPS mice originated from alveolar type II cells and lung macrophages, as demonstrated by immunofluorescent staining. Dysfunctional vessel formation in CBDL mice was visualized by microscopy on vascular corrosion casts. Both prophylactic and therapeutic anti-PlGF (aPlGF) antibody treatment impeded HPS development, as demonstrated by significantly less intrapulmonary shunting and improved gas exchange. aPlGF treatment decreased endothelial cell dysfunction in vivo and in vitro and was accompanied by reduced pulmonary inflammation. Importantly, aPlGF therapy did not affect liver alterations, supporting aPlGF's ability to directly target the pulmonary compartment. Conclusion: CBDL in mice induces HPS, which is mediated by PlGF production; aPlGF treatment improves experimental HPS by counteracting pulmonary angiogenesis and might be an attractive therapeutic strategy for human HPS. (HEPATOLOGY 2018; 68:634-651) 

H epatopulmonary syndrome (HPS) refers to a clinical triad: (1) impaired arterial oxygenation, (2) pulmonary microvascular alterations, and (3) occurrence in a setting of liver disease. (1) This complication develops in up to 30% of patients with cirrhosis and, if left untreated, carries a poor prognosis. (2) Despite growing knowledge of the mechanisms involved in HPS development, its pathogenesis Abbreviations: aPlGF, anti-PlGF; AT2, alveolar type 2; BALF, bronchoalveolar lavage fluid; CBDL, common bile duct ligation; CD68, cluster of differentiation 68; ELISA, enzyme-linked immunosorbent assay; ENG, endoglin; G-CSF, granulocyte-colony stimulating factor; HPS, hepatopulmonary syndrome; IgG, immunoglobulin G1; IL, interleukin; KC, keratinocyte chemoattractant; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein 1; NE, neutrophil elastase; PaO 2 , partial pressure of arterial oxygen; PlGF, placental growth factor; SEM, scanning electron microscopy; VCAM-1, vascular cell adhesion molecule 1; VEGF, vascular endothelial growth factor; VEGFR1/2, vascular endothelial growth factor receptors 1/2; vWF, von Willebrand factor.

has not been fully unraveled. No effective medical therapies are available, and liver transplantation remains the only curative option. However, successful transplantation is often hampered by progressive hypoxemia, (3) further highlighting the need for discovering new treatments.

Rat studies have shown that endothelin-1-mediated vasodilation, (4) (5) (6) (7) bacterial translocation, (8) (9) (10) (11) and angiogenesis (12) (13) (14) are key components in HPS pathogenesis. Recent reports have also described involvement of the respiratory epithelial compartment, (15, 16) which needs further investigation. Extensive evidence supports angiogenesis as a crucial mechanism in microvascular proliferation and intrapulmonary shunt formation, which is responsible for gas exchange impairment in HPS. Angiogenesis refers to new blood vessel formation from preexisting vessels, an event that is mediated by angiogenic growth factors. Preclinical studies in common bile duct ligation (CBDL) rats demonstrated that multitargeted antiangiogenic therapies improve experimental HPS. (13, 14) This resulted in an ongoing randomized clinical trial that is studying the multi-tyrosine kinase inhibitor sorafenib as treatment for HPS (ClinicalTrials.gov, NCT02021929). Although multitargeted therapies may be successful, their clinical use is often limited by disabling adverse effects, especially in fragile patients including patients with cirrhosis and complications such as HPS. Thus, for these patients there is still an important unmet medical need.

The development of novel therapies is based on preclinical studies using established animal models covering the pathognomonic features of human disease. At present, CBDL in rats is the only reported animal model for HPS that mimics the pulmonary vascular abnormalities of human HPS. (17) However, in-depth investigation of pathogenic mechanisms relies on the use of transgenic models and antibody-mediated inhibition studies which require a mouse model. As the development of pulmonary angiogenesis during the onset and progression of biliary liver fibrosis in mice has not been outlined yet, we first aimed to validate CBDL in mice as a model for human HPS by focusing on impaired gas exchange and pulmonary vascular alterations during the kinetics of HPS development. Placental growth factor (PlGF) is a key proangiogenic molecule and a member of the vascular endothelial growth factor (VEGF) family and exclusively regulates angiogenesis in pathological conditions. (18) PlGF specifically binds to VEGF receptor 1 (VEGFR1), its soluble form, and the coreceptors neuropilin 1/2. (19) PlGF stimulates endothelial cell growth, migration, and survival in two ways: (1) indirectly by displacement of VEGF from VEGFR1, thereby triggering VEGF/VEGFR2 signaling pathways and (2) directly by VEGFR1 activation and stimulation of crosstalk between VEGFR1 and VEGFR2. (19) In addition, anti-PlGF (aPlGF) antibodies have been shown to efficiently and selectively inhibit pathological angiogenesis (18, 20) with a favorable safety profile (20) and without the typical side effects seen with other antiangiogenic regimens. Moreover, most angiogenic inhibitors encounter the development of resistance to therapy based on an escape mechanism through "switch on" of compensatory proangiogenic programs. (19) Besides producing a prosurvival effect on endothelial cells, PlGF also attracts VEGFR1-expressing macrophages and bone marrow progenitors, which further stimulate the angiogenic process by additional growth factor release. (19) Unlike other antiangiogenic agents, aPlGF therapy has been shown to prevent infiltration with angiocompetent macrophages, which might abolish this antiangiogenic escape. (19, 20) As such, aPlGF might represent an attractive tool in the therapy for diseases that are mediated by excessive angiogenesis, including HPS.

In the current work, we first validated a mouse model for HPS, evaluated the contribution of PlGF in this model, and then explored its potential as therapeutic target.
Materials and Methods

ANIMALS
Seven-week-old male Swiss mice (Harlan, Brussels, Belgium) were housed in open cages in a temperaturecontrolled room at 20 8C with a 12 hour dark/12 hour light cycle at the animal facility of the Faculty of Medicine and Health Sciences (Ghent University, Belgium). Animals had free access to water and commercial chow (mouse maintenance chow; Carfil Labofood, Belgium). Mice were acclimatized under controlled conditions for 1 week prior to CBDL or sham surgery, which were performed as described (21) (Supporting Information). All mice received care in accordance with the Guide for the Care and Use of Laboratory Animals and the Belgian national guidelines for animal protection. The Ethical Committee of Experimental Animals at the faculty of Medicine and Health Science, Ghent University, Belgium, approved the experiments (ECD 14/67).
KINETIC STUDY ON DEVELOPMENT OF HEPATIC FIBROSIS AND PULMONARY ANGIOGENESIS AND INFLAMMATION
To investigate hepatic and pulmonary angiogenic and inflammatory changes during the development of biliary fibrosis and HPS following CBDL, mice were sacrificed on a weekly basis for 6 consecutive weeks, starting 1 week postsurgery (n 5 6-14 animals per group). At sacrifice, serum, bronchoalveolar lavage fluid (BALF), lungs, liver, and spleen were collected for analysis. In a separate experiment, the pulmonary vascular network of sham-operated (n 5 3) and CBDL mice with cirrhosis (n 5 3) was visualized by scanning electron microscopy (SEM) on vascular corrosion casts.
DETERMINATION OF INTRAPULMONARY SHUNT FORMATION
The degree of intrapulmonary shunting using microspheres was assessed by a modification of the methods described by Fallon et al. (17) and Miniati et al. (22) Sham and CBDL mice were anesthetized with pentobarbital (intraperitoneally, 40 mg/kg, Nembutal; Ceva Sant e Animale, Brussels, Belgium), and the inferior caval vein and carotid artery were isolated. Next, 1 3 10 6 fluorescent polystyrene microspheres (6 mm diameter; Phosphorex Inc., Hopkinton, MA) in phosphate-buffered saline were injected into the inferior caval vein. Immediately after injection of the microspheres, the carotid artery was transected and the exsanguinated blood recovered. Fluorescence intensity of the arterial blood was measured on a spectrophotometer (Tecan Safire II, data analysis software Magellan 6; Tecan Trading AG, Switzerland) and corresponds to the degree of intrapulmonary shunting, after correction for collected blood volume.
PlGF INHIBITION STUDIES
Anti-PlGF neutralizing monoclonal antibodies (5D11D4; ThromboGenics NV, Leuven, Belgium) were tested for their potential to inhibit pathological angiogenesis and gas exchange impairment in CBDLinduced mice, in a preventive and in a therapeutic setting (n 5 4-8 animals per group). Sham and CBDL mice received intraperitoneal antibody administration twice weekly, at a dose of 25 mg/kg (concentration 5.4 mg/mL, volume injected 140-185 mL). A group of matched sham and CBDL mice were injected with mouse immunoglobulin G1 (IgG; ThromboGenics NV) as control at the same dose and times as aPlGFtreated mice (concentration 4.46 mg/mL, volume injected 110-225 mL). The dosing schedule of aPlGF was based on previous pharmacokinetic studies in mice. (20, 23) In the preventive study, treatment was administered starting at the day of surgery and continued until week 6; in the therapeutic study, the treatment regimen was started at the beginning of postsurgery week 2. Mice were sacrificed 6 weeks postsurgery, and arterial blood, serum, BALF, lungs, liver, and spleen were collected for analysis. In a separate experiment, SEM was performed on lung vascular corrosion casts of CBDL mice that were preventively treated with IgG or aPlGF (n 5 6 animals).
HUMAN SAMPLES
Serum levels of endoglin (ENG) were assessed by a Luminex magnetic bead assay in blood samples from patients with cirrhosis and HPS (n 5 30) and without HPS (n 5 30). Additional information regarding laboratory technique and demographic and clinical characteristics of the patients included in the study can be found in the Supporting Information.
STATISTICAL ANALYSIS
Statistical analyses were performed using GraphPad Prism software version 6.0 (GraphPad, La Jolla, CA) and SPSS Statistics version 23 (SPSS Inc., Chicago, IL). Data distribution was evaluated with the Shapiro-Wilk test. Normally distributed data were analyzed with the Student t test or analysis of variance. In cases of non-normal distributions, data were analyzed using the Mann-Whitney U or Kruskal-Wallis test. Two-tailed probabilities were calculated, and P values < 0.05 were considered significant.
STATISTICAL ANALYSIS
Additional information on the surgical procedures, arterial blood gas analysis, bronchoalveolar lavage and cytospins, vascular corrosion casting, histology, myeloperoxidase assay, enzyme-linked immunosorbent assay (ELISA), multiplex magnetic bead technology, western blot analysis, cell culture, in vitro experiments, and human samples can be found in the Supporting Information.
Results

VALIDATION OF THE HPS MOUSE MODEL AND KINETICS OF PULMONARY ANGIOGENESIS AND INFLAMMATION

Clinicopathological Features of Liver Injury After CBDL
To establish an HPS mouse model, we used CBDL, which is an archetype model for obstructive cholestasis, liver fibrosis, and portal hypertension in rats and mice. CBDL resulted in increased liver and spleen weights starting from week 1 and continuing until week 6 and decreased body weight at week 6 postsurgery compared to sham mice (Supporting Table S2 ). Liver histology after CBDL showed bile duct proliferation, while no morphological changes in sham controls were observed. Liver fibrosis increased from an F0-1 METAVIR score at week 1 to F4 at week 6 after CBDL (Supporting Fig. S1 ). No significant increases in protein expression of PlGF, VEGF, or VEGFR1/2 could be demonstrated in hepatic tissue of CBDL mice compared to sham mice (Supporting Fig. S2 ).
CBDL Mice Exhibit Hypoxemia and Intrapulmonary Shunting, in Accordance With HPS
To validate CBDL in mice as an experimental HPS model, gas exchange impairment and the presence of intrapulmonary shunting were assessed. At 6 weeks postsurgery, CBDL mice suffered from significant hypoxemia as demonstrated by arterial blood gas analysis (mean partial pressure of arterial oxygen [PaO 2 ] 63.2 6 9.2 and 92.2 6 6.7 mm Hg in CBDL and sham, respectively; P 5 .007, Fig. 1A ). The presence of intrapulmonary shunting was evaluated using intravenous microsphere injections. Because the diameter of the normal mouse pulmonary microvasculature measures <6 mm, most of the microsphere beads injected into the inferior caval vein are trapped in the lung. In HPS, microspheres are able to pass through intrapulmonary vascular dilations and shunts, and the beads can be detected in the arterial blood. Intrapulmonary shunting developed from week 1 after CBDL (P 5 0.017 versus sham) and further aggravated until week 6 (P 5 0.0045 versus sham) (Fig. 1A) . These observations at 6 weeks post-CBDL in conjunction with biliary liver damage and cirrhosis (Supporting Fig. S1 ) fulfill the clinical criteria for HPS diagnosis and validate this mouse model for HPS. Architectural changes of the pulmonary vasculature were further investigated by microscopic evaluation of lung vascular casts. Lungs of mice with cirrhosis displayed a chaotically disorganized pulmonary vascular network, marked by tortuosity, scattered lumpy regions, and global distortion of the normally well-organized honeycomb-like capillary network seen in sham mice (Fig. 1B) , consistent with previous studies. (24, 25) 
Development of HPS Is Characterized by Pulmonary Angiogenesis EXPERIMENTAL HPS INDUCTION ACTI-VATES THE PULMONARY ENDOTHELIUM, WHICH CORRESPONDS TO HUMAN HPS BIOMARKERS
We and others have previously reported on elevated circulating vascular cellular adhesion molecule 1 (VCAM-1) and von Willebrand factor (vWF) in HPS patients, indicating that these factors may be promising biomarkers for early detection of HPS. (26, 27) To extend these findings, we measured serum levels of ENG, a third marker of endothelial cell activation, in the same patient cohort. In accordance, ENG serum levels had significantly increased in HPS patients compared to non-HPS patients with cirrhosis (mean 6.1 6 0.6 versus 4.1 6 0.4 ng/mL; P 5 0.007; Fig.  1C ). To directly associate these data with pulmonary angiogenesis, we took advantage of our mouse model and analyzed vWF and ENG immunoreactivity in lungs of CBDL and sham mice. As shown by vWFimmunopositive and ENG-immunopositive microvessels, pulmonary angiogenic spots were observed in CBDL mice from week 4 (P 5 0.05 versus sham) and progressively increased until week 6 (P 5 0.0009 versus sham) (Fig. 1D) . VCAM-1 levels were measured in CBDL mice sera because specifically soluble VCAM-1, shed by the endothelium in case of sustained endothelial activation, exerts a proangiogenic function. (28) Analogous to the human condition, significantly elevated serum levels were observed in CBDL mice, from week 1 (P 5 0.0048 versus sham) and continuing until week 6 (P 5 0.0028 versus sham) (Fig. 1E) .
PULMONARY ANGIOGENESIS IN EXPERI-MENTAL HPS IS MARKED BY AN UP-REGULATION OF PLGF, WHICH IS PRODUCED BY ALVEOLAR TYPE II CELLS AND LUNG MACROPHAGES
To investigate the involvement of PlGF-mediated signaling in pulmonary angiogenesis, angiogenic markers of the VEGF family were measured at regular intervals following surgery. CBDL led to significant up-regulation of PlGF in the lungs from week 2 (P 5 0.016 versus sham) to week 6 (P 5 0.0019 versus sham) postinduction ( Fig. 2A) , while no increase in circulating PlGF could be observed (data not shown) comparable to the human situation. (26) PlGF colocalized with cluster of differentiation 68 (CD68; monocyte/macrophage marker) and thyroid transcription factor-1 (a nuclear alveolar type II [AT2] cell marker) ( Fig. 2B-D) ; this finding implies that lung macrophages and AT2 cells are the source of PlGF in the lungs of HPS mice. No substantial up-regulation of VEGF or VEGFR1/2 expression in lung tissue of CBDL mice could be demonstrated ( Fig. 2A) . A significant increase in circulating soluble VEGFR1 was detected from week 1 (P 5 0.0012 versus sham) and continued until week 6 (P < 0.0001 versus sham) postsurgery ( Fig. 2A) .
Pulmonary Angiogenesis in HPS Development Is Accompanied by Pulmonary Inflammation
Because PlGF has been linked to angiogenesisassociated inflammation in various pathologies, we determined the time frame in which proinflammatory factors are up-regulated in HPS mice lungs. Increased levels of monocyte chemoattractant protein 1 (MCP-1), which is the main mediator for monocyte attraction to the target site, were observed from week 1 (MCP-1, P 5 0.001 versus sham) post-CBDL until endpoint (MCP-1, P 5 0.0045) (Fig.  F3  3A) . This was accompanied by an increase, which was most pronounced during the earlier stages of HPS development, in keratinocyte chemoattractant (KC), which is responsible for neutrophil migration, and interleukin 1b (IL-1b) (Fig. 3A) . To investigate leukocyte infiltration, we analyzed pulmonary myeloperoxidase levels, which is an enzyme present in neutrophil granules and monocyte lysosomes, and performed cytological analysis with BALF samples. CBDL caused a significant increase in pulmonary myeloperoxidase activity (Fig.  3B ) and the absolute numbers of neutrophils in BALF compared to sham controls (Fig. 3C ). In line with this, levels of neutrophil elastase (NE), which is a protease produced by activated neutrophils that is responsible for breakdown of the alveolar epithelial compartment, were elevated in BALF of CBDL mice in comparison to sham at weeks 5 (P 5 0.044) and 6 (P < 0.0001) (Fig. 3D) .
PlGF INHIBITION AMELIORATES CBDL-INDUCED HPS BY COUNTERACTING PULMONARY ANGIOGENESIS

Anti-PlGF Antibodies Are Able to Prevent HPS Development in CBDL Mice
To first assess the role of PlGF in HPS development, CBDL and sham mice were treated with aPlGF antibodies on the day of surgery and continued until week 6. Treatment with aPlGF was well tolerated in sham and CBDL animals, and body weight was similar in mice treated with aPlGF and IgG (Supporting Table S3 ). Importantly, prophylactic aPlGF treatment was able to prevent pulmonary gas exchange defects following CBDL as observed in IgG-treated CBDL mice (mean PaO 2 90.5 6 6.2 versus 63.2 6 9.2 mm Hg; P 5 0.04; Fig. 4A ). This was confirmed by lower arterial fluorescence intensity following intravenous microsphere injection as a measure for intrapulmonary shunt formation (P 5 0.01; Fig. 4A ) and partial normalization of the distorted, densely packed pulmonary microvessels as observed by SEM in aPlGF-injected compared to IgGinjected CBDL mice (Fig. 4B) .
HPS in Mice Can Be Reversed by aPlGF Treatment
As a next step, we were interested in the therapeutic potential of aPlGF therapy for treating established HPS. Anti-PlGF antibodies were administered starting at the beginning of week 2 post-CBDL, when intrapulmonary shunting is known to be advanced (Fig. 1A) . Administration of aPlGF to HPS mice resulted in improved pulmonary gas exchange (mean PaO 2 90.1 6 6.5 mm Hg in aPlGF versus 63.4 6 7.5 mm Hg in IgG-treated mice; P 5 0.03), with reduction of intrapulmonary shunting (P 5 0.01) (Fig.  5A) . The treatment regimen was well tolerated, and no significant effects on body weight were observed (Supporting Table S3 ).
PlGF Inhibition Improves HPS Through Pulmonary Endothelial Normalization Without Induction of a Rescue Angiogenic Program
Based on the obtained results, we hypothesized that aPlGF's beneficial effect on experimental HPS could be attributed to modulation of pulmonary endothelial dysfunction. Lungs of IgG-treated CBDL mice were marked by scattered vWF-immunopositive and ENGimmunopositive endothelial spots (preventive vWF, P < 0.0001; ENG, P < 0.0001 versus IgG sham, Fig.  4C ; therapeutic setting vWF, P 5 0.0003; ENG, P 5 0.0004 versus IgG sham; Fig. 5B ), whereas CBDL mice that received aPlGF showed decreased vWF (P 5 0.003 and P 5 0.0008 versus IgG CBDL) and ENG (P 5 0.0008 and P 5 0.01 versus IgG CBDL mice) (Figs. 4C and 5B), which is indicative of reduced endothelial cell activation. This was accompanied by a trend toward lower circulating VCAM-1 in aPlGF-treated compared to IgG-treated CBDL mice (preventive, P 5 0.09; therapeutic, P 5 0.36; Figs. 4D and 5C).
PlGF Inhibition Improves HPS Through Pulmonary Endothelial Normalization Without Induction of a Rescue Angiogenic Program
Because antiangiogenic regimens are known for their potential to induce a rescue angiogenic program, we measured the expression of other proangiogenic member molecules of the VEGF family. Anti-PlGF antibody administration did not induce compensatory expression of VEGF or VEGFR1/2, indicating a negligible induction of angiogenic escape mechanisms (Figs. 4D and 5C ).
PlGF Inhibition Attenuates Pulmonary Inflammation
Because angiogenesis and inflammation are closely linked, we were interested in whether PlGF activity suppression and the associated endothelial normalization also affected pulmonary inflammation in HPS. Anti-PlGF treatment resulted in a marked reduction in pulmonary MCP-1 expression (preventive, P 5 0.06; therapeutic, P 5 0.01 versus IgG-treated CBDL; Figs. 4E and 5D), which was the only chemokine that was up-regulated at end-stage fibrosis with HPS. Because MCP-1 mediates the attraction of monocytes/macrophages to the target site, we assessed pulmonary CD68 expression. In sham animals, CD68 levels were minimal, while a significant increase was seen in CBDL mice (preventive, sham versus CBDL IgG, P 5 0.0001; therapeutic, sham versus CBDL IgG, P 5 0.0276) (Figs. 4F and 5E ). Anti-PlGFtreated animals showed reduced CD68 expression compared with untreated CBDL animals (preventive, P 5 0.0028; therapeutic, P 5 0.05) (Figs. 4F and 5E) , with lower macrophage histological counts (hematoxylin and eosin sections, preventive, P 5 0.0084 and therapeutic, P 5 0.033; CD68 sections, preventive, P 5 0.0021 and therapeutic, P 5 0.24) (Supporting Fig. S3 ). In addition, NE levels in BALF were lower (although not significant) in aPlGF-treated CBDL mice compared to IgG-treated animals (preventive, P 5 0.067; therapeutic, P 5 0.071) (Supporting Fig.  S4 ).
Modulation of PlGF Signaling Modifies Endothelial Cell, Macrophage, and AT2 Cell Activation in Response to Lipopolysaccharide In Vitro
In vitro experiments were designed to further explore the behavior of the cells lining the alveolar septum. Lipopolysaccharide (LPS) was selected as the stimulant because bacterial translocation associated with liver disease is seen as the initial trigger for HPS development. (29) To verify the normalizing effect of aPlGF on the endothelium, MS1 endothelial cells were stimulated with LPS in the presence or absence of aPlGF. Incubation with LPS alone induced secretion of MCP-1, KC, and granulocyte colonystimulating factor (G-CSF) (all P < 0.0001) and VCAM-1 expression (P 5 0.0047), which was significantly less pronounced during aPlGF cotreatment (Fig. 6A) .
Modulation of PlGF Signaling Modifies Endothelial Cell, Macrophage, and AT2 Cell Activation in Response to Lipopolysaccharide In Vitro
In parallel, bone marrow-derived macrophages stimulated with LPS secreted MCP-1, KC, G-CSF (all P < 0.0001), and IL-1b (P 5 0.005). However, addition of aPlGF to cell medium only resulted in less MCP-1 secretion (P 5 0.026) compared to LPS challenge only (Fig. 6B) .
Modulation of PlGF Signaling Modifies Endothelial Cell, Macrophage, and AT2 Cell Activation in Response to Lipopolysaccharide In Vitro
MLE-12 alveolar epithelial cells stimulated with LPS showed elevated MCP-1 (P < 0.0001), KC (P 5 0.0003), and G-CSF (P 5 0.0015) secretion, along with increased caspase 3/7 activity (P 5 0.0078), which was inhibited by the addition of aPlGF (MCP-1, P 5 0.0148; KC, P 5 0.082; G-CSF, P 5 0.019; caspase 3/7, P 5 0.023) (Fig. 6C) .
PlGF INHIBITION DOES NOT AFFECT THE LIVER FOLLOWING CBDL
We and others have reported on the therapeutic effects of PlGF blockade on liver fibrosis in CCl 4 -induced mice. (30, 31) Although HPS develops on a background of liver fibrosis, this could only be confirmed following CBDL. (12) To investigate whether aPlGF's therapeutic effects on HPS and pulmonary alterations are due to reduced liver disease in CBDL mice, we compared the degree of liver fibrosis and hepatic expression of proangiogenic and inflammatory markers in CBDL mice treated with either aPlGF or IgG. CBDL IgG-treated animals showed significantly increased hepatic fibrosis compared to sham controls (preventive, P 5 0.0004; therapeutic, P < 0.0001; Fig.  7A ), without any difference after aPlGF treatment (preventive, P 5 0.5; therapeutic, P 5 0.36) (Fig. 7A) . Although hemodynamic measurements were not performed, histological evaluation of the mesentery showed no effect of PlGF blockade on the splanchnic vascular bed (Supporting Fig. S5 ), and spleen enlargement, a surrogate indicator of portal hypertension, was unaltered following aPlGF treatment (preventive, 0.23 6 0.030 versus 0.23 6 0.043 g; P 5 0.95; therapeutic, 0.32 6 0.034 versus 0.25 6 0.030 g; P 5 0.16) (Supporting Table S3 ). MCP-1 and KC levels were markedly elevated in hepatic tissue of CBDL Data are represented as means 6 SE. *P < 0.05, **P < 0.01, ***P < 0.001.
FIG. 6.
Effects of aPlGF on endothelial cell, macrophage, and AT2 cell response to LPS challenge in vitro. MS1 endothelial cells, bone marrow-derived macrophages, and MLE-12 lung epithelial cells were stimulated with LPS to mimic bacterial translocation associated with liver disease. (A) VCAM-1 expression (ELISA) and MCP-1, KC, and G-CSF levels (multiplex magnetic bead technology) in supernatant from unstimulated and LPS-stimulated MS1 cells with or without aPlGF. (B) MCP-1, KC, G-CSF, and IL-1b levels (multiplex bead-based technology) in supernatant from unstimulated and LPS-stimulated bone marrow-derived macrophages with or without aPlGF. (C) MCP-1, KC, and G-CSF levels (multiplex bead-based technology) in supernatant and caspase 3/7 activity in unstimulated and LPS-stimulated MLE-12 cells with or without aPlGF. Data are represented as means 6 SE. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: BMDM, bone marrow-derived macrophage; NS, not significant. compared to sham mice and remained unaffected after aPlGF treatment (MCP-1 preventive, P 5 0.38; therapeutic, P 5 0.61; KC preventive, P 5 0.44; therapeutic, P 5 0.38) (Fig. 7C,E) . Irrespective of the treatment regimen, neither PlGF, VEGF, nor VEGFR1/2 expressions were up-regulated in CBDL mice livers (Fig. 7B,D) .
Discussion
Multiple lines of evidence support that pulmonary angiogenesis serves as a principal event in experimental HPS development. (8, 12, 32) However, reports are limited to observations in rats, and the pathogenesis of pulmonary microvascular alterations in HPS remains poorly understood, which contributes to the lack of effective medical therapies. In this article, we describe the establishment of an HPS mouse model, the role of PlGF in HPS development, and the efficacy of aPlGF treatment for HPS in this model.
Discussion
Several animal models of liver disease have been evaluated for their potential to mimic HPS. So far, only CBDL in rats has recapitulated human HPS. (12) Our first aim was to establish an HPS mouse model in order to expand the possibility of future research on HPS pathogenesis and development of new therapeutic agents. We have documented that CBDL in mice, as in rats, induces HPS. CBDL mice develop intrapulmonary shunting and become hypoxemic on a background of cholestatic liver disease, which fulfills the three diagnostic criteria of HPS.
Discussion
(1) To our knowledge, only one study has reported on CBDL in mice as a potential model for HPS, which, however, failed to demonstrate intrapulmonary shunts due to methodological issues. (33) Our findings are in line with knowledge from human patients (1, 34) and prior work in rats, (8, 12, 17) in which it was shown that shunts start to develop early after CBDL induction and do not require advanced fibrosis. We and others have shown that endothelial dysfunction markers in serum might predict HPS in patients with cirrhosis. (26, 27) Our current observation that circulating ENG levels, similar to vWF and VCAM-1, are increased in this same cohort of HPS patients further adds to the concept of enhanced angiogenesis as a pivotal contributor to HPS development. To verify if these specific factors are of actual importance in HPS pathogenesis, their role was assessed in the established HPS mouse model. Pulmonary endothelium of HPS mice specifically expressed vWF and ENG as scattered angiogenic spots and secreted VCAM-1. These events become aggravated over the course of HPS development and are indicative of progressing endothelial dysfunction. The similarities between experimentally induced and human HPS hereby confirm that the CBDL mouse model qualifies as a valuable model to further investigate pathological angiogenesis in the pathogenesis of HPS and will be valuable to test novel treatments.
Discussion
Although intrapulmonary vasodilation, angiogenesis, and inflammation have been identified to play a key role in HPS pathogenesis, clinical studies targeting these pathways in the past were rather disappointing. (34) At present, sorafenib is being tested as a therapeutic strategy for HPS, but important questions arise with respect to its safety because multitargeted therapies are notorious for their associated toxicities. In this regard, we hypothesized that PlGF, a proangiogenic molecule with restricted activity in pathological conditions, could serve as a potential target for HPS. We have shown that PlGF is up-regulated in CBDL mouse lungs early during HPS development and that PlGF is locally produced by intrapulmonary macrophages and AT2 cells. Our study presents evidence concerning the capacity of aPlGF therapy to prevent and reverse experimental HPS in mice with improved gas exchange, decreased intrapulmonary shunting, and attenuation of endothelial activation and pulmonary angiogenesis. Down-regulation of pulmonary vWF and ENG, partial normalization of the threedimensional structure of the pulmonary vascular network, and amelioration of HPS clinical signs following aPlGF administration further support a direct role for PlGF in HPS and associated pulmonary angiogenesis.
Discussion
The therapeutic potential of PlGF blockade has been a subject of study in different pathologies, most of which are related to ophthalmology and oncology. Anti-PlGF antibodies have been shown to selectively inhibit pathological angiogenesis, without affecting healthy vessels and thereby causing minimal side effects, and have been demonstrated to be safe (20) and well-tolerated in human healthy volunteers and oncologic patients. (35) (36) (37) Previous studies have revealed that PlGF is dispensable for physiological vessel maintenance in healthy mice while contributing to the angiogenic switch in case of pathology. (19, 20) Importantly, aPlGF treatment did not alter expression of VEGF family members in the present study, indicating minimal induction of angiogenic escape.
Discussion
In vitro experiments further support the concept of aPlGF's normalizing effect on the endothelium as the main underlying mechanism of HPS improvement. This concept was demonstrated by a reduction of VCAM-1 and proinflammatory signature of endothelial cells when subjected to LPS stimulation; these findings are consistent with our in vivo observations. Our results further indicated an additional effect on macrophage MCP-1 secretion, which was reduced upon PlGF inhibition. Indeed, aPlGF treatment in vivo resulted in decreased expression of proinflammatory markers and CD68 levels. This is a valuable finding because bacterial translocation with subsequent pulmonary inflammation is assumed to be the main driver of HPS onset (10, 11, 29, 38, 39) and might contribute to antiangiogenic escape. (19, 20) Later stages in HPS development in our study were characterized by pulmonary neutrophil infiltration and elevated levels of NE in BALF. Other studies have described a role for elastase and PlGF in inducing AT2 cell death during pulmonary emphysema, (40) (41) (42) and a recent report demonstrated AT2 cell dysfunction after CBDL in rats. (16) In line with this, we have shown that aPlGF attenuates the AT2 cell proinflammatory and caspase 3/7 response to LPS in vitro. However, targeting the AT2 cell itself as a novel therapeutic option in HPS warrants further investigation.
Discussion
Finally, we and others have reported on the potential of PlGF as a therapeutic target in chronic liver disease using the CCl 4 model. (30, 31) Based on these results, one could postulate that the beneficial effects of aPlGF on HPS originate from a favorable primary effect on the liver. However, PlGF was not up-regulated in liver tissue of CBDL mice, and no differences in hepatic fibrosis, spleen weight, and inflammatory markers could be demonstrated between aPlGF-treated and IgG-treated CBDL mice. This finding supports aPlGF's capability to limit HPS through a direct effect on the pulmonary compartment. Even though CCl 4 and CBDL are two highly reliable models sharing a common outcome (liver fibrosis), their pathogenic mechanisms are far from comparable. CCl 4 leads to centrilobular hepatocyte death and pericentral fibrosis, whereas CBDL results in obstructive cholestasis and bile duct proliferation with portal inflammation and periportal fibrosis. (21) These dissimilarities might explain the differences in hepatic PlGF expression between different animal models and explain why HPS only develops in CBDL-induced mice.
Discussion
In conclusion, we validated a mouse model for HPS and identified PlGF as an important contributor to pulmonary alterations in HPS pathogenesis. Our data provide evidence for aPlGF antibodies as a therapeutic strategy against HPS, a life-threatening disorder for which no effective medical treatment is available at this time.

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