M2 macrophage accumulation contributes to pulmonary fibrosis, vascular dilatation, and hypoxemia in rat hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) markedly increases the mortality of patients. However, its pathogenesis remains incompletely understood. Rat HPS develops in common bile duct ligation (CBDL)-induced, but not thioacetamide (TAA)-induced cirrhosis. We investigated the mechanisms of HPS by comparing these two models. Pulmonary histology, blood gas exchange, and the related signals regulating macrophage accumulation were assessed in CBDL and TAA rats. Anti-polymorphonuclear leukocyte (antiPMN) and anti-granulocyte-macrophage colony stimulating factor (antiGM-CSF) antibodies, clodronate liposomes (CL), and monocyte chemoattractant protein 1 (MCP1) inhibitor (bindarit) were administrated in CBDL rats, GM-CSF, and MCP1 were administrated in bone marrow-derived macrophages (BMDMs). Pulmonary inflammatory cell recruitment, vascular dilatation, and hypoxemia were progressively developed by 1 week after CBDL, but only occurred at 4 week after TAA. Neutrophils were the primary inflammatory cells within 3 weeks after CBDL and at 4 week after TAA. M2 macrophages were the primary inflammatory cells, meantime, pulmonary fibrosis, GM-CSFR, and CCR2 were specifically increased from 4 week after CBDL. AntiPMN antibody treatment decreased neutrophil and macrophage accumulation, CL or the combination of antiGM-CSF antibody and bindarit treatment decreased macrophage recruitment, resulting in pulmonary fibrosis, vascular dilatation, and hypoxemia in CBDL rats alleviated. The combination treatment of GM-CSF and MCP1 promoted cell migration, M2 macrophage differentiation, and transforming growth factor-ß1 (TGF-ß1) production in BMDMs. Conclusively, our results highlight neutrophil recruitment mediates pulmonary vascular dilatation and hypoxemia in the early stage of rat HPS. Further, M2 macrophage accumulation induced by GM-CSF/GM-CSFR and MCP1/CCR2 leads to pulmonary fibrosis and promotes vascular dilatation and hypoxemia, as a result, HPS develops.

Tea polyphenols and Levofloxacin alleviate the lung injury of hepatopulmonary syndrome in common bile duct ligation rats through Endotoxin -TNF signaling.

BACKGROUND & AIMS: Hepatopulmonary syndrome (HPS) is characterized by pulmonary vasodilation and arterial blood oxygen desaturation in patients with chronic liver disease. Generally, common bile duct ligation (CBDL) rats are a suitable experimental model for studying hepatopulmonary syndrome. Our previous study demonstrated that endotoxin surges markedly, followed by bacterial translocation and the loss of liver immune function in all the stages of CBDL, thereby contributing to the pathogenesis of HPS. However, the mechanisms behind the increase of the endotoxin and how to alleviate it have not yet been elucidated. Pulmonary injury induced by increased bilirubin, endotoxin, and inflammatory mediators occurs in the early and later stages of CBDL. This study assessed the effects of Tea polyphenols (TP) and Levofloxacin on endotoxin reduction and suppression of lung injury in HPS rats in the long and short term, respectively. METHODS: Morphological change of pulmonary injury, HPS relative index, endotoxin concentration, and the activation extent of Malondialdehyde (MDA) and Myeloperoxidase (MPO) were evaluated in CBDL rats with or without TP and Levofloxacin for three weeks or six weeks. The inflammation factors of serum, lung tissue, and BALF were then compared at the same condition for the two time periods. This was followed by adoption of the network pharmacology approach, which was mainly composed of active component gathering, target prediction, HPS gene collection, network analysis, and gene enrichment analysis. Finally, the mRNA and protein levels of the inflammatory factors were studied and relative signaling expression was assessed using RT-PCR and Western blot analysis. RESULTS: The obtained results indicated that the pulmonary injury manifestation was perceived and endotoxin, MDA, and MPO activation were markedly increased in the early and later stages of CBDL. TP and Levofloxacin treatment alleviated endotoxin infection and inflammation factor expression three weeks and six weeks after CBDL. In addition, Levofloxacin displayed a short time anti-bacterial effect, while TP exerted a long period function. TP and Levofloxacin also reduced TNF-α, TGF-ß, IL-1ß, PDGF-BB, NO, ICAM-1, and ET-1 expression on the mRNA or protein expression. With regard to the pharmacological mechanism, the network analysis indicated that 12 targets might be the therapeutic targets of TP and Levofloxacin on HPS, namely ET-1, NOs3, VEGFa, CCl2, TNF, Ptgs2, Hmox1, Alb, Ace, Cav1, and Mmp9. The gene enrichment analysis implied that TP and Levofloxacin probably benefited patients with HPS by modulating pathways associated with the AGE-RAGE signaling pathway, the TNF signaling pathway, the HIF-1 signaling pathway, the VEGF signaling pathway, and the IL-17 signaling pathway, Rheumatoid arthritis, Fluid shear stress, and atherosclerosis. Finally, the TNF-α level was mainly diminished on the protein level following CBDL. CONCLUSIONS: TP and Levofloxacin could alleviate pulmonary injury for short and long period, respectively, while at the same time preventing endotoxin and the development of HPS in CBDL rats. These effects are possibly associated with the regulation of the Endotoxin -TNF-α pathways.

Diosmin enhances the anti-angiogenic activity of sildenafil and pentoxifylline against hepatopulmonary syndrome via regulation of TNF-α/VEGF, IGF-1/PI3K/AKT, and FGF-1/ANG-2 signaling pathways.

Hepatopulmonary syndrome (HPS) is a severe complication of hepatic cirrhosis, which is characterized by hypoxia, intrapulmonary vasodilation, inflammation, and angiogenesis. In this study, we aimed to investigate the regulatory effects of diosmin (DS) on selected phosphodiesterase inhibitors against chronic bile duct ligation (CBDL)-induced HPS. Experimentally, Wistar Albino rats were used and HPS was induced by CBDL for 28 days. DS (100 mg/kg, daily, P.O.), sildenafil (Sild; 10 mg/kg, twice daily, P.O.), and pentoxifylline (PTX; 50 mg/kg, daily, P.O.) were evaluated either alone or in combinations for their anti-angiogenic activity. CBDL significantly altered oxidative stress biomarkers and up-regulated pulmonary mRNA expressions of VEGF, IGF-1, ET-1, iNOS, eNOS, and ANG-2 as well as the protein expressions of vWF, FGF-1, PI3K, AKT, p-AKT, TGF-ß, HYP, MPO activity and circulating TNF-α. Treatment with DS, Sild, PTX, and their combinations significantly attenuated molecular and cellular changes due to CBDL. Improvement of histopathological changes was also observed after drug treatment which further supported our results. Furthermore, DS combination with Sild or PTX exhibited an improvement in HPS in comparison to each drug alone. Collectively, DS can augment the anti-angiogenic activity of Sild and PTX during HPS through regulation of TNF-α/VEGF, IGF-1/PI3K/AKT, and FGF-1/ANG-2 signaling pathways.

Impact of liver damage on blood-borne variables and pulmonary hemodynamic responses to hypoxia and hyperoxia in anesthetized rats.

BACKGROUND: Liver disorders may be associated with normal pulmonary hemodynamic, hepatopulmonary syndrome (HPS), or portopulmonary hypertension (POPH). In this study, we aimed to investigate the effect of the severity of liver dysfunctions on blood-borne variables, and pulmonary hemodynamic during repeated ventilation with hyperoxic and hypoxic gases. METHODS: Female Sprague Dawley rats were assigned into four groups of Sham (n = 7), portal vein ligation (PPVL, n = 7), common bile duct ligation (CBDL, n = 7), and combination of them (CBDL+ PPVL, n = 7). Twenty-eight days later, right ventricular systolic pressure (RVSP) and systemic blood pressure were recorded in anesthetized animals subjected to repeated maneuvers of hyperoxia (O2 50%) and hypoxia (O2 10%). Besides, we assessed blood parameters and liver histology. RESULTS: Liver histology score, liver enzymes, WBC and plasma malondialdehyde in the CBDL+PPVL group were higher than those in the CBDL group. Also, the plasma platelet level in the CBDL+PPVL group was lower than those in the other groups. On the other hand, the serum estradiol in the CBDL group was higher than that in the CBDL+PPVL group. All the above parameters in the PPVL group were similar to those in the Sham group. During ventilation with hyperoxia gas, RVSP in the CBDL+PPVL group was higher than the ones in the other groups, and in the CBDL group, it was more than those in the PPVL and Sham groups. Hypoxic pulmonary vasoconstriction (HPV) was not detected in both CBDL+PPVL and CBDL groups, whereas, it retained in the PPVL group. CONCLUSION: Severe liver damage increases RVSP in the CBDL+PPVL group linked to the high level of ROS, low levels of serum estradiol and platelets or a combination of them. Furthermore, the high RVSP at the noted group could present a reliable animal model for POPH in female rats.

CX3CR1 participates in pulmonary angiogenesis in experimental hepatopulmonary syndrome mice through inhibiting AKT/ERK signaling pathway and regulating NO/NOS release.

OBJECTIVE: Hepatopulmonary syndrome (HPS) is a kind of pulmonary microvascular disease and occurs in 15%-30% cirrhosis. This study aimed to investigate the effects of pulmonary CX3CR1 on angiogenesis and associated mechanisms in HPS animal models. MATERIALS AND METHODS: CX3CR1GFP/GFP mice were constructed by replacing CX3CR1 with GFP. Common bile duct ligation (CBDL) mouse model was established with surgery. Release of nitric oxide (NO) was evaluated. Hematoxylin-eosin (HE) staining was employed to examine the inflammation of lung tissues. CD31 expression was detected with immunohistochemistry assay. Western blotting was used to evaluate the expression of CX3CL1, CX3CR1, phosphorylated-AKT (p-AKT), phosphorylated-ERK (p-ERK). Quantitative Real Time-PCR (qRT-PCR) assay was used to examine VEGF, PDGF, iNOS, eNOS, and HO-1 expression. RESULTS: CX3CR1-deficiency (CX3CR1GFP/GFP-sham or CX3CR1GFP/GFP-CBDL mice) significantly reduced NO release compared to wide type (WT)-mice or WT-CBDL mice (p<0.05). CX3CR1-deficiency significantly alleviated inflammation compared to wide type (WT)-mice or WT-CBDL mice (p<0.05). CX3CR1-deficiency significantly reduced CD31 expression compared to WT-sham and WT-CBDL mice, respectively (p<0.05). CX3CR1 also participated in anti-angiogenesis efficacy of Bevacizumab. CX3CR1-deficiency significantly down-regulated the ratio of p-AKT/AKT and p-ERK/ERK and inhibited the secretion of VEGF and PDGF compared to WT-mice (p<0.05). CX3CR1-deficiency significantly reduced iNOS, eNOS, and HO-1 expression compared to WT-mice (p<0.05). CONCLUSIONS: CX3CR1 deficiency reduced VEGF and PDGF production, inhibited p-AKT, and p-ERK activation and down-regulated iNOS, eNOS, and HO-1 expression. Therefore, CX3CR1 participates in pulmonary angiogenesis in the experimental HPS mice via inhibiting AKT/ERK signaling pathway and regulating NO/NOS release. These findings would provide a potential insight for clarifying the pathological mechanisms of HPS.

Loss of cell polarity regulated by PTEN/Cdc42 enrolled in the process of Hepatopulmonary Syndrome.

One central factor in hepatopulmonary syndrome (HPS) pathogenesis is pulmonary vascular remodelling (PVR) which involves dysregulation of proliferation and migration in pulmonary microvascular endothelial cells (PMVECs). Growing evidence suggests that Apical/basolateral polarity plays an important role in cell proliferation, migration, adhesion and differentiation. In this study, we explored whether cell polarity is involved and critical in experimental HPS rats that are induced by common bile duct ligation (CBDL). Cell polarity related proteins were analysed in CBDL rats lung and PMVECs under the HPS serum stimulation by immunofluorescence assay. Cdc42/PTEN activity, cell proliferation and migration and Annexin A2 (AX2) in PMVECs were determined, respectively. Cell polarity related proteins, lost their specialized luminal localization in PMVECs of the CBDL rat. The loss of cell polarity was induced by abnormal activity of Cdc42, which was strongly enhanced by the interaction between p-PTEN and Annexin A2 in PMVECs, after treatment with serum from CBDL rats. It led to over-proliferation and high migration ability of PMVECs. Down-regulation of PTEN-Cdc42 activity in PMVECs restored cell polarity and thus reduced their ability of migration and proliferation. Our study suggested that the loss of cell polarity plays a critical role in the pathogenesis of HPS-associated PVR and may become a potentially effective therapeutic target.

Effects of Caffeine Treatment on Hepatopulmonary Syndrome in Biliary Cirrhotic Rats.

Hepatopulmonary syndrome (HPS) is a lethal complication of cirrhosis characterized by hypoxia and overt intrapulmonary shunting. In this study, we investigated the effect of caffeine in rats with common bile duct ligation (CBDL)-induced liver cirrhosis and HPS. CBDL rats were randomly allocated to receive caffeine or vehicle for 14 days. On the 28th day after CBDL, mortality rate, hemodynamics, liver, and renal biochemistry parameters and arterial blood gas analysis were evaluated. Lung and liver were dissected for the evaluation of inflammation, angiogenesis and protein expressions. In another series with parallel groups, the intrapulmonary shunting was determined. Caffeine significantly reduced portal pressure (caffeine vs. control: 10.0 ± 3.7 vs. 17.0 ± 8.1 mmHg, p < 0.05) in CBDL rats. The mortality rate, mean arterial pressure, biochemistry data and hypoxia were similar between caffeine-treated and control groups. Caffeine alleviated liver fibrosis and intrahepatic angiogenesis but intrapulmonary inflammation and angiogenesis were not ameliorated. The hepatic VEGF/Rho-A protein expressions were down-regulated but the pulmonary inflammation- and angiogenesis-related protein expressions were not significantly altered by caffeine. Caffeine did not reduce the intrapulmonary shunting, either. Caffeine has been shown to significantly improve liver fibrosis, intrahepatic angiogenesis and portal hypertension in cirrhotic rats, however, it does not ameliorate HPS.

Melatonin effects on pulmonary tissue in the experimental model of Hepatopulmonary Syndrome / Efeitos da melatonina sobre o tecido pulmonar no modelo experimental de Síndrome Hepatopulmonar

ABSTRACT Objective: To evaluate the pulmonary alterations of animals with Hepatopulmonary Syndrome (HPS) submitted to Biliary Duct Ligature (BDL), as well as the antioxidant effect of Melatonin (MEL). Methods: Sixteen male Wistar rats, divided into four Sham groups: BDL group, Sham + MEL group and BDL + MEL. The pulmonary and hepatic histology, lipoperoxidation and antioxidant activity of lung tissue, alveolar-arterial O2 difference and lung / body weight ratio (%) were evaluated. Results: When comparing the groups, could be observed an increase of vasodilation and pulmonary fibrosis in the BDL group and the reduction of this in relation to the BDL + MEL group. It was also observed significant changes in the activity of catalase, ApCO2, ApO2 in the LBD group when compared to the other groups. Conclusion: The use of MEL has been shown to be effective in reducing vasodilation, fibrosis levels and oxidative stress as well as gas exchange in an experimental HPS model.

RESUMO Objetivo: Avaliar as alterações pulmonares de animais com Síndrome Hepatopulmonar (SHP), submetidos à ligadura de ducto biliar (LDB), bem como o efeito antioxidante da Melatonina (MEL). Métodos: Dezesseis ratos machos da espécie Wistar, divididos em quatro grupos: Sham, Grupo LDB, Grupo Sham + MEL e LDB + MEL. Foram avaliadas a histologia pulmonar e hepática, a lipoperoxidação e atividade antioxidante do tecido pulmonar, diferença álveolo-arterial de O2 e relação peso pulmonar/peso corporal (%). Resultados: Quando comparados os grupos, observamos um aumento da vasodilatação e fibrose pulmonar no grupo LDB e a redução deste em relação ao grupo LDB+MEL. Observamos ainda alterações significativas na atividade da catalase, PaCO2, PaO2 no grupo LBD quando comparado aos demais grupos. Conclusões: A utilização da MEL demonstrou-se eficaz na redução da vasodilatação, níveis de fibrose e estresse oxidativo assim como na troca gasosa em modelo experimental de SHP.

miR144-3p inhibits PMVECs excessive proliferation in angiogenesis of hepatopulmonary syndrome via Tie2.

BACKGROUND/AIM: Increasing evidence show microRNAs (miRNAs) are associated with hepatopulmonary syndrome (HPS). The aim of this study was to investigate the role of miR-144 in the angiogenesis of HPS, as well as to identify its underlying mechanism. METHODS: The expression levels of miR-144-3p were assessed in pulmonary micro-vascular endothelial cells (PMVECs), as well as in lung tissues from rats with HPS. We predicted the potential target of miR-144-3p. Tyrosine kinase 2(Tie2) was identified as a target gene of miR144-3p, which has an essential role in the angiogenesis of lung vessel. In addition, the effects of miR-144-3p regulated on Tie2 was examined. The upregulation and down-regulation of miR-144-3p can affect the proliferation of PMVECs. RESULTS: We found that the levels of miR-144-3p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-144-3p dramatically inhibited PMVECs proliferation and cell cycle. We further verified the Tie2 as a novel and direct target of miR-144-3p in HPS. CONCLUSION: miR-144-3p can negatively regulate PMVECs proliferation by Tie2 expression. In addition, overexpression of miR-144-3p may prove beneficial as a therapeutic strategy for HPS treatment.

AMD3100 treatment attenuates pulmonary angiogenesis by reducing the c-kit (+) cells and its pro-angiogenic activity in CBDL rat lungs.

Recent studies have shown that pulmonary angiogenesis is an important pathological process in the development of hepatopulmonary syndrome (HPS), and growing evidence has indicated that Stromal cell-derived factor 1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) axis is involved in pulmonary vascular disease by mediating the accumulation of c-kit+ cells. This study aimed to test the effect of AMD3100, an antagonist of CXCR4, in HPS pulmonary angiogenesis. Common bile duct ligation (CBDL) rats were used as experimental HPS model and were treated with AMD3100 (1.25mg/kg/day, i.p.) or 0.9% saline for 3weeks. The sham rats underwent common bile duct exposure without ligation. The c-kit+ cells accounts and its angiogenic-related functions, prosurvival signals, pulmonary angiogenesis and arterial oxygenation were analysed in these groups. Our results showed that pulmonary SDF-1/CXCR4, Akt, Erk and VEGF/VEGFR2 were significantly activated in CBDL rats, and the numbers of circulating and pulmonary c-kit+ cells were increased in CBDL rats compared with control rats. Additionally, the angiogenic-related functions of c-kit+ cells and pulmonary microvessel counts were also elevated in CBDL rats. CXCR4 inhibition reduced pulmonary c-kit+ cells and microvessel counts and improved arterial oxygenation within 3weeks in CBDL rats. The pulmonary prosurvival signals and pro-angiogenic activity of c-kit+ cells were also down-regulated in AMD3100-treated rats. In conclusion, AMD3100 treatment attenuated pulmonary angiogenesis in CBDL rats and prevented the development of HPS via reductions in pulmonary c-kit+ cells and inhibition of the prosurvival signals. Our study provides new insights in HPS treatment.

The ET-1-mediated carbonylation and degradation of ANXA1 induce inflammatory phenotype and proliferation of pulmonary artery smooth muscle cells in HPS.

Hepatopulmonary syndrome (HPS) is a serious complication of advanced liver disease, which markedly increases mortality. Pulmonary vascular remodelling (PVR) induced by circulating mediators plays an important role in the pathogenesis of HPS, while the underlying mechanism remains undefined. In the present study, we reported that endothelin-1 (ET-1) is up-regulated and annexin A1(ANXA1) is down-regulated in HPS rat, and ET-1 decreases the ANXA1 expression in a dose-dependent manner in rat pulmonary arterial smooth muscle cells (PASMCs). Then, we showed that ANXA1 can decrease nuclear p-ERK1/2 accumulation and decrease the cyclin D1 expression, thus resulting in the subsequent inhibition of PASMCs proliferation. As previously reported, we confirmed that ET-1 decreases the ANXA1 protein levels by the carbonylation and degradation of ANXA1. In conclusion, our research links the signaling cascade of ET1-ANXA1-cell proliferation to a potential therapeutic strategy for blocking IPS-associated PVR.

Proteomics-based analysis of lung injury-induced proteins in a mouse model of common bile duct ligation.

BACKGROUND: Lung injury is a life-threatening complication in patients with liver dysfunction. We recently provided an experimental lung injury model in mouse with common bile duct ligation. In this study, we aimed to characterize the pathologic and biochemical features of lung tissues in common bile duct ligation mice using a proteomic approach. METHODS: Common bile ducts of BALB/c mice, 8 weeks of age, were ligated operatively. CD31-expressing pulmonary cells were sorted with immunomagnetic microbeads, and protein profiles were examined by 2-dimensional gel electrophoresis. Based on the results of protein identification, immunohistochemistry and quantitative reverse transcription polymerase chain reaction were carried out in pulmonary and hepatic tissues. RESULTS: Two-dimensional gel electrophoresis revealed 3 major inflammation-associated proteins exhibiting considerable increases in the number of CD31-positive pulmonary cells after common bile duct ligation. Mass spectrometry analysis identified these proteins as SerpinB1a (48 kDa), ANXA1 (46 kDa), and S100A9 (16 kDa). Furthermore, the 3 proteins were more highly expressed in dilated pulmonary blood vessels of common bile duct ligation mice, in which neutrophils and monocytes were prominent, as shown by immunohistochemistry. More importantly, SerpinB1a mRNA and protein were significantly upregulated in the liver, whereas S100A9 and ANXA1 mRNA and protein were upregulated in the lungs, as shown by quantitative reverse transcription polymerase chain reaction and Western blotting. CONCLUSION: We identified 3 proteins that were highly expressed in the lung after common bile duct ligation using a proteomics-based approach.

Quercetin alleviates pulmonary angiogenesis in a rat model of hepatopulmonary syndrome.

Quercetin shows protective effects against hepatopulmonary syndrome (HPS), as demonstrated in a rat model. However, whether these effects involve pulmonary vascular angiogenesis in HPS remains unclear. Therefore, this study aimed to assess the effect of quercetin on pulmonary vascular angiogenesis and explore the underlying mechanisms. Male Sprague-Dawley rats weighing 200-250 g underwent sham operation or common bile duct ligation (CBDL). Two weeks after surgery, HIF-1α and NFκB levels were assessed in rat lung tissue by immunohistochemistry and western blot. Then, CBDL and sham-operated rats were further divided into 2 subgroups each to receive intraperitoneal administration of quercetin (50 mg/kg daily) or 0.2% Tween for two weeks: Sham (Sham+Tween; n=8), CBDL (CBDL+Tween; n=8), Q (Sham+quercetin; n=8), and CBDL+Q (CBDL+quercetin; n=8). After treatment, lung tissue specimens were assessed for protein (immunohistochemistry and western blot) and/or gene expression (quantitative real-time PCR) levels of relevant disease markers, including VEGFA, VEGFR2, Akt/p-Akt, HIF-1α, vWf, and IκB/p-IκB. Finally, arterial blood was analyzed for alveolar arterial oxygen pressure gradient (AaPO2). Two weeks after CBDL, HIF-1α expression in the lung decreased, but was gradually restored at four weeks. Treatment with quercetin did not significantly alter HIF-1α levels, but did reduce AaPO2 as well as lung tissue NF-κB activity, VEGFA gene and protein levels, Akt activity, and angiogenesis. Although hypoxia is an important feature in HPS, our findings suggest that HIF-1α was not the main cause for the VEGFA increase. Interestingly, quercetin inhibited pulmonary vascular angiogenesis in rats with HPS, with involvement of Akt/NF-κB and VEGFA/VEGFR-2 pathways.

Role of splenic reservoir monocytes in pulmonary vascular monocyte accumulation in experimental hepatopulmonary syndrome.

BACKGROUND AND AIM: Pulmonary monocyte infiltration plays a significant role in the development of angiogenesis in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). Hepatic monocytes are also increased after CBDL, but the origins remain unclear. Splenic reservoir monocytes have been identified as a major source of monocytes that accumulate in injured tissues. Whether splenic monocytes contribute to monocyte alterations after CBDL is unknown. This study evaluates monocyte distributions and assesses effects of splenectomy on monocyte levels and pulmonary vascular and hepatic abnormalities in experimental HPS. METHODS: Splenectomy was performed in CBDL animals. Monocyte levels in different tissues and circulation were assessed with CD68. Pulmonary alterations of HPS were evaluated with vascular endothelial growth factor-A (VEGF-A) levels, angiogenesis, and alveolar-arterial oxygen gradient (AaPO2 ). Liver abnormalities were evaluated with fibrosis (Sirius red), bile duct proliferation (CK-19), and enzymatic changes. RESULTS: Monocyte levels increased in the lung and liver after CBDL and were accompanied by elevated circulating monocyte numbers. Splenectomy significantly decreased monocyte accumulation, VEGF-A levels, and angiogenesis in CBDL animal lung and improved AaPO2 levels. In contrast, hepatic monocyte levels, fibrosis, and functional abnormalities were further exacerbated by spleen removal. CONCLUSIONS: Splenic reservoir monocytes are a major source for lung monocyte accumulation after CBDL, and spleen removal attenuates the development of experimental HPS. Liver monocytes may have different origins, and accumulation is exacerbated after depletion of splenic reservoir monocytes. Tissue specific monocyte alterations, influenced by the spleen reservoir, have a significant impact on pulmonary complications of liver disease.

Annexin A2 inhibits the migration of PASMCs stimulated with HPS rat serum by down-regulating the expression of paxillin.

Hepatopulmonary syndrome (HPS) has been classically associated with intrapulmonary vasodilatation (IPVD) and pulmonary vascular remodelling (PVR), which are the key pathophysiological components of HPS and concerned frequently in the studies of HPS. Little is known about the relevance of pulmonary artery smooth muscle cells (PASMCs) migration or the molecular mechanisms of PVR in HPS. Annexin A2 (ANXA2) plays crucial role in HPS-associated PVR and might activate the activity of paxillin which as a regulatory protein participates in the regulation of PASMCs function in PVR. In addition, it has been identified that ANXA2 could influence the cells migration by some important signaling pathways in many diseases, including lung cancer, pulmonary hypertensionand and liver cancer. In this study, we performed scratch wound motility assay, modified boyden chamber, reverse transcription PCR, western blot and co-immunoprecipitation to determine the role of ANXA2 on HPS-associated PVR. We found that HPS rat serum from a common bile duct ligation (CBDL) rat model enhanced the migration of PASMCs and increased the expression of ANXA2 in PASMCs. We reported that ANXA2 and paxillin could form a co-immunoprecipitation. After silencing ANXA2 with siRNA, we found that the up-regulation of paxillin expression, induced by the HPS rat serum, was reversed. Additionally, we found that down-regulation of ANXA2 could significantly inhibit the migration of PASMCs. These findings indicated that down-regulation of ANXA2 by siRNA results in the inhibition of the aberrant dysregulation of paxillin and migration of PASMCs, which suggesting a potential therapeutic effect on HPS-associated PVR.

Quercetin alleviates pulmonary angiogenesis in a rat model of hepatopulmonary syndrome

Quercetin shows protective effects against hepatopulmonary syndrome (HPS), as demonstrated in a rat model. However, whether these effects involve pulmonary vascular angiogenesis in HPS remains unclear. Therefore, this study aimed to assess the effect of quercetin on pulmonary vascular angiogenesis and explore the underlying mechanisms. Male Sprague-Dawley rats weighing 200-250 g underwent sham operation or common bile duct ligation (CBDL). Two weeks after surgery, HIF-1α and NFκB levels were assessed in rat lung tissue by immunohistochemistry and western blot. Then, CBDL and sham-operated rats were further divided into 2 subgroups each to receive intraperitoneal administration of quercetin (50 mg/kg daily) or 0.2% Tween for two weeks: Sham (Sham+Tween; n=8), CBDL (CBDL+Tween; n=8), Q (Sham+quercetin; n=8), and CBDL+Q (CBDL+quercetin; n=8). After treatment, lung tissue specimens were assessed for protein (immunohistochemistry and western blot) and/or gene expression (quantitative real-time PCR) levels of relevant disease markers, including VEGFA, VEGFR2, Akt/p-Akt, HIF-1α, vWf, and IκB/p-IκB. Finally, arterial blood was analyzed for alveolar arterial oxygen pressure gradient (AaPO2). Two weeks after CBDL, HIF-1α expression in the lung decreased, but was gradually restored at four weeks. Treatment with quercetin did not significantly alter HIF-1α levels, but did reduce AaPO2 as well as lung tissue NF-κB activity, VEGFA gene and protein levels, Akt activity, and angiogenesis. Although hypoxia is an important feature in HPS, our findings suggest that HIF-1α was not the main cause for the VEGFA increase. Interestingly, quercetin inhibited pulmonary vascular angiogenesis in rats with HPS, with involvement of Akt/NF-κB and VEGFA/VEGFR-2 pathways.

Effect of the oestrogen receptor antagonist fulvestrant on the cirrhotic rat lung.

It has been postulated that cirrhosis-related lung vasodilatation and the subsequent hepatopulmonary syndrome are partly explained by an increased estradiol level through an enhanced endothelial formation of nitric oxide (NO). In this study, we assessed whether the oestrogen receptor antagonist fulvestrant (F) improves cirrhosis-related lung abnormalities. Cirrhosis was induced in rats by chronic bile duct ligation (CBDL). Four groups were studied: CBDL, CBDL+F, sham, and sham+F. Histological, immunohistochemical, and Western blot analyses were performed on lung samples. In the lung, the endothelial NO synthase and the nitrotyrosine protein expressions were increased in CBDL as compared to sham rats. Both parameters were significantly reduced by fulvestrant in the CBDL rats. Surprisingly, the level of pVASP (an indirect marker of NO formation and action) was decreased in CBDL rats, and fulvestrant had no effect on this parameter. The level of the vascular endothelial growth factor, the diameter of small lung vessels, and the number of macrophages were increased in CBDL lungs in comparison with sham lungs, and these parameters were unaffected by fulvestrant treatment. In conclusion, fulvestrant may not be relevant to improve lung abnormalities in cirrhosis because NO may not be biologically active and because key events contributing to the lung abnormalities are not affected by fulvestrant.

Caspase-3 inhibition prevents the development of hepatopulmonary syndrome in common bile duct ligation rats by alleviating pulmonary injury.

BACKGROUND & AIMS: Common bile duct ligation (CBDL) rats is an accepted experimental model of hepatopulmonary syndrome (HPS), defined as liver disease and intrapulmonary vascular dilatation and hypoxaemia. Pulmonary Akt and ERK activation followed by angiogenesis in the later stages of CBDL, contribute to the pathogenesis of HPS. However, the mechanisms behind Akt and ERK activation remain undefined. Pulmonary injury induced by increased bilirubin, endotoxin and inflammatory mediators occurs in the early stages of CBDL. We assessed the effects of relieving pulmonary injury on Akt and ERK activation and on the development of HPS following CBDL. METHODS: Pulmonary injury, angiogenesis, arterial oxygenation, cell proliferation and, phospho-Akt and ERK1 were evaluated in CBDL animals with or without caspase-3 inhibition (Z-DEVD-FMK). Pulmonary injury was assessed by histology and quantifying apoptosis and aquaporin-1 (AQP1) levels. Lung angiogenesis was assessed by quantifying AQP1 level, vWF-positive cells and microvessel count. RESULTS: Pulmonary apoptosis and caspase-3 activation were markedly increased in the early stages of CBDL. Caspase-3 inhibition alleviated apoptosis, the reduction in AQP1, phospho-Akt and ERK1 levels and pulmonary injury 1 week after CBDL. Caspase-3 inhibition also reduced AQP1, phospho-Akt and ERK1 levels, vWF-positive cells, cell proliferation, microvessel count, and microvascular dilatation and improved arterial oxygenation 3 weeks following CBDL. CONCLUSIONS: Caspase-3 inhibition alleviates pulmonary injury, thereby preventing angiogenesis as well as the development of HPS in CBDL rats. These effects are related to the regulation of the Akt and ERK1 pathways.

Alveolar type II epithelial cell dysfunction in rat experimental hepatopulmonary syndrome (HPS).

The hepatopulmonary syndrome (HPS) develops when pulmonary vasodilatation leads to abnormal gas exchange. However, in human HPS, restrictive ventilatory defects are also observed supporting that the alveolar epithelial compartment may also be affected. Alveolar type II epithelial cells (AT2) play a critical role in maintaining the alveolar compartment by producing four surfactant proteins (SPs, SP-A, SP-B, SP-C and SP-D) which also facilitate alveolar repair following injury. However, no studies have evaluated the alveolar epithelial compartment in experimental HPS. In this study, we evaluated the alveolar epithelial compartment and particularly AT2 cells in experimental HPS induced by common bile duct ligation (CBDL). We found a significant reduction in pulmonary SP production associated with increased apoptosis in AT2 cells after CBDL relative to controls. Lung morphology showed decreased mean alveolar chord length and lung volumes in CBDL animals that were not seen in control models supporting a selective reduction of alveolar airspace. Furthermore, we found that administration of TNF-α, the bile acid, chenodeoxycholic acid, and FXR nuclear receptor activation (GW4064) induced apoptosis and impaired SP-B and SP-C production in alveolar epithelial cells in vitro. These results imply that AT2 cell dysfunction occurs in experimental HPS and is associated with alterations in the alveolar epithelial compartment. Our findings support a novel contributing mechanism in experimental HPS that may be relevant to humans and a potential therapeutic target.

[Protective effect of tanshinol on the hepatopulmonary syndrome in rat].

OBJECTIVE: To explore the mechanism of tanshinol on alleviate the inflammatory injury of lung tissue in rat hepatopulmonary syndrome (HPS). METHODS: SD rats were randomly divided into normal control group (n = 8), hepatopulmonary syndrome (HPS) group (n = 11) and tanshinol intervention group (n = 9). HE staining was used to observe the histopathology changes of pulmonary and hepatic tissues, and to count the number of macrophages in lung tissues. The activity of alanine transferase (ALT) and concentrations of endotoxin, tumor necrosis factor-a (TNF-alpha) and homocystein (Hcy) in plasma were detected. The concentrations of TNF-alpha, nitric oxide (NO) and malondialdehyde (MDA) and the activity of inducible nitric oxide synthase (iNOS) in the lung tissues were measured, respectively. RESULTS: Thickened alveolar septum and increased macrophages were observed in lungs in HPS rat. After administered with tanshinol, the pulmonary pathological changes were alleviated and the number of macrophages in lung tissue was decreased compared with HPS group. The activity of ALT and the concentrations of endotoxin, TNF-alpha and Hcy in plasma ,and TNF-alpha, iNOS, NO and MDA in lung tissue in HPS group were higher than those of normal control group; meanwhile, those tanshinol group were less those that of HPS group. CONCLUSION: Tanshinol may play an important role in delaying the development of HPS through protecting liver or directly antagonizing the effect of intestinal endotoxemia so as to alleviate the inflammatory reaction in lung tissue.