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.

Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling.

Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling.

MicroRNA-199a-5p Regulates the Proliferation of Pulmonary Microvascular Endothelial Cells in Hepatopulmonary Syndrome.

BACKGROUND/AIMS: Pulmonary microvascular endothelial cell (PMVEC) proliferation and angiogenesis contribute to the development of hepatopulmonary syndrome (HPS). MicroRNA-199a-5p (miR-199a-5p) has emerged as a potent regulator of angiogenesis, and its expression levels significantly decrease in the serum of patients with hepatopathy. However, it has not been reported about whether miR-199a-5p might control PMVEC proliferation. Here, we described the miR-199a-5p governing PMVEC proliferation in HPS. METHODS: PMVECs were treated with rat serum from common bile duct ligation (CBDL) or sham. MiR-199a-5p mimic or inhibitor was used to change the miR-199a-5p expression. Knockdown of caveolin-1 (Cav-1) was performed using siRNA. NSC-23766 was used to inhibit Rac1 activity. Gene and protein expressions were quantified by qRT-PCR and western blot. Cell proliferation was analyzed by 3H-TdR incorporation and CCK-8 assays. Stress fibers were detected by immunofluorescence. RESULTS: CBDL rat serum induced the down-regulation of miR-199a-5p. Delivery of miR-199a-5p suppressed the CBDL rat serum-induced PMVEC proliferation whereas knockdown of miR-199a-5p promoted PMVEC proliferation. This was accompanied by a decrease and an increase in Cav-1 expression, respectively. Cav-1 siRNA abolished the enhancement of PMVEC proliferation induced by the miR-199a-5p inhibition. Although stress fibers were disrupted in Cav-1 deficient cells, NSC-23766 increased stress fibers and contributed to cell proliferation. CONCLUSIONS: CBDL rat serum induced down-regulation of miR-199a-5p in PMVECs, which led to an increase of Cav-1 gene expression. Increased Cav-1 expression, by inhibiting Rac1 activity, led to the formation of stress fibers, which contribute to PMVEC proliferation and thus the pathogenesis of HPS.

Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2.

Aberrant constitutive expression of c-Rel, p65 and p50 NF-kappaB subunits has been reported in over 90% of breast cancers. Recently, we characterized a de novo RelB NF-kappaB subunit synthesis pathway, induced by the cytomegalovirus (CMV) IE1 protein, in which binding of p50-p65 NF-kappaB and c-Jun-Fra-2 AP-1 complexes to the RELB promoter work in synergy to potently activate transcription. Although RelB complexes were observed in mouse mammary tumours induced by either ectopic c-Rel expression or carcinogen exposure, little is known about RelB in human breast disease. Here, we demonstrate constitutive de novo RelB synthesis is selectively active in invasive oestrogen receptor alpha (ERalpha)-negative breast cancer cells. ERalpha signalling reduced levels of functional NF-kappaB and Fra-2 AP-1 and inhibited de novo RelB synthesis, leading to an inverse correlation between RELB and ERalpha gene expression in human breast cancer tissues and cell lines. Induction of Bcl-2 by RelB promoted the more invasive phenotype of ERalpha-negative cancer cells. Thus, inhibition of de novo RelB synthesis represents a new mechanism whereby ERalpha controls epithelial to mesenchymal transition (EMT).

The Therapeutic Effects of Baicalein on the Hepatopulmonary Syndrome in the Rat Model of Chronic Common Bile Duct Ligation.

Background and Aims: Hepatopulmonary syndrome (HPS) is characterized by arterial oxygenation defects due to pulmonary vascular dilation in liver disease. To date, liver transplantation remains the only effective treatment for HPS. This study aimed to explore the preventative role of baicalein in HPS development. Methods: Sixty male rats were randomly assigned to three groups: sham, common bile duct ligation (CBDL), and baicalein, receiving intraperitoneal injections of baicalein (40 mg·kg-1·d-1, diluted in saline) for 21 days. Survival rate, liver and kidney function, and bile acid metabolism levels were evaluated. Liver and lung angiogenesis and hepatic glycogen staining were assessed, and the expression of relevant proteins was evaluated by immunohistochemistry. Results: Baicalein improved survival rates and hypoxemia in rats post-CBDL, reducing angiogenic protein levels and enhancing glucose homeostasis. Compared to the untreated group, baicalein suppressed the expression of vascular endothelial growth factor, placental growth factors, matrix metalloprotease 9 and C-X-C motif chemokine 2, and it increased the expression of glycemic regulatory proteins, including dipeptidyl peptidase-4, sirtuin 1, peroxisome proliferator-activated receptor gamma co-activator 1α, and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3. Conclusion: Baicalein significantly improves hepatic function and hypoxia in HPS rats by attenuating pathological angiogenesis in the liver and lungs, showing promise as a treatment for HPS.

Basal actomyosin pulses expand epithelium coordinating cell flattening and tissue elongation.

Actomyosin networks constrict cell area and junctions to alter cell and tissue shape. However, during cell expansion under mechanical stress, actomyosin networks are strengthened and polarized to relax stress. Thus, cells face a conflicting situation between the enhanced actomyosin contractile properties and the expansion behaviour of the cell or tissue. To address this paradoxical situation, we study late Drosophila oogenesis and reveal an unusual epithelial expansion wave behaviour. Mechanistically, Rac1 and Rho1 integrate basal pulsatile actomyosin networks with ruffles and focal adhesions to increase and then stabilize basal area of epithelial cells allowing their flattening and elongation. This epithelial expansion behaviour bridges cell changes to oocyte growth and extension, while oocyte growth in turn deforms the epithelium to drive cell spreading. Basal pulsatile actomyosin networks exhibit non-contractile mechanics, non-linear structures and F-actin/Myosin-II spatiotemporal signal separation, implicating unreported expanding properties. Biophysical modelling incorporating these expanding properties well simulates epithelial cell expansion waves. Our work thus highlights actomyosin expanding properties as a key mechanism driving tissue morphogenesis.

Extrahepatic Angiogenesis: A Potential Common Pathophysiological Basis of Multiple Organ Dysfunction in Rats with Cholestasis Cirrhosis.

BACKGROUND: In addition to intrahepatic angiogenesis, patients with cholestasis cirrhosis develop extrahepatic vasculature disorders and functional disturbances of multiple organ systems. Without effective intervention, these vascular disorders will eventually turn into multiple organs vascular syndromes, including the brain, lung and other organ systems. However, studies on the pathogenesis of vascular alterations among extrahepatic organ disturbances are still carried out separately, which hampered the successful translation of preclinical studies to the human setting and required further mechanistic insight into these complications. This study aims to investigate the relationship between extrahepatic angiogenesis and multiple organ impairment, and whether the vascular endothelial growth factor (VEGF) family members and their receptors are involved in this process. METHODS: Pathological changes of the multiple organs were determined by histopathological and immunohistochemical staining in the established common bile duct ligation (CBDL) rats, and angiogenesis was estimated by microvessel density (MVD). Levels of the VEGF family members and their receptors in the serum and organ tissues were also measured by using enzyme-linked immunosorbent assays. RESULTS: The MVD and VEGF family members and their receptors were significantly increased in CBDL rats with multiple organ injury, especially in the liver, lung and cerebral cortex. Meanwhile, we noticed moderate elevation of soluble receptor of the vascular endothelial growth factor-1 (sFlt-1) in the liver, lung, and cerebral cortex, whereas the levels of placental growth factor (PLGF) increased significantly. CONCLUSIONS: Extrahepatic angiogenesis may represent a common pathophysiological basis for multiple organ dysfunction and the sFlt-1/PLGF ratio could offer an avenue for further studies to target extrahepatic angiogenesis in cholestatic cirrhosis.

Serum Soluble Vascular Endothelial Growth Factor Receptor 1 as a Potential Biomarker of Hepatopulmonary Syndrome.

Background and Aims: The results of basic research implicate the vascular endothelial growth factor (VEGF) family as a potential target of hepatopulmonary syndrome (HPS). However, the negative results of anti-angiogenetic therapy in clinical studies have highlighted the need for markers for HPS. Therefore, we aimed to determine whether VEGF family members and their receptors can be potential biomarkers for HPS through clinical and experimental studies. Methods: Clinically, patients with chronic liver disease from two medical centers were enrolled and examined for HPS. Patients were divided into HPS, intrapulmonary vascular dilation [positive contrast-enhanced echocardiography (CEE) and normal oxygenation] and CEE-negative groups. Baseline information and perioperative clinical data were compared between HPS and non-HPS patients. Serum levels of VEGF family members and their receptors were measured. In parallel, HPS rats were established by common bile duct ligation. Liver, lung and serum samples were collected for the evaluation of pathophysiologic changes, as well as the expression levels of the above factors. Results: In HPS rats, all VEGF family members and their receptors underwent significant changes; however, only soluble VEGFR1 (sFlt-1) and the sFlt-1/ placental growth factor (PLGF) ratio were changed in almost the same manner as those in HPS patients. Furthermore, through feature selection and internal and external validation, sFlt-1 and the sFlt-1/PLGF ratio were identified as the most important variables to distinguish HPS from non-HPS patients. Conclusions: Our results from animal and human studies indicate that sFlt-1 and the sFlt-1/PLGF ratio in serum are potential markers for HPS.

Two Rac1 pools integrate the direction and coordination of collective cell migration.

Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, here we reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, our studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.

PKCθ-mediated serine/threonine phosphorylations of FAK govern adhesion and protrusion dynamics within the lamellipodia of migrating breast cancer cells.

The cytoskeleton and cell-matrix adhesions constitute a dynamic network that controls cellular behavior during development and cancer. The Focal Adhesion Kinase (FAK) is a central actor of these cell dynamics, promoting cell-matrix adhesion turnover and active membrane fluctuations. However, the initial steps leading to FAK activation and subsequent promotion of cell dynamics remain elusive. Here, we report that the serine/threonine kinase PKCθ participates in the initial steps of FAK activation. PKCθ, which is strongly expressed in aggressive human breast cancers, controls the dynamics of cell-matrix adhesions and active protrusions through direct FAK activation, thereby promoting cell invasion and lung metastases. Using various tools for in vitro and live cell studies, we precisely decipher the molecular mechanisms of FAK activation. PKCθ directly interacts with the FAK FERM domain to open FAK conformation through PKCθ's specific V3 domain, while phosphorylating FAK at newly identified serine/threonine residues within nascent adhesions, inducing cell dynamics and aggressive behavior. This study thus places PKCθ-directed FAK opening and phosphorylations as an original mechanism controlling dynamic, migratory, and invasive abilities of aggressive breast cancer cells, further strengthening the emerging oncogenic function of PKCθ.

The Emerging Function of PKCtheta in Cancer.

Protein Kinase C theta (PKCθ) is a serine/threonine kinase that belongs to the novel PKC subfamily. In normal tissue, its expression is restricted to skeletal muscle cells, platelets and T lymphocytes in which PKCθ controls several essential cellular processes such as survival, proliferation and differentiation. Particularly, PKCθ has been extensively studied for its role in the immune system where its translocation to the immunological synapse plays a critical role in T cell activation. Beyond its physiological role in immune responses, increasing evidence implicates PKCθ in the pathology of various diseases, especially autoimmune disorders and cancers. In this review, we discuss the implication of PKCθ in various types of cancers and the PKCθ-mediated signaling events controlling cancer initiation and progression. In these types of cancers, the high PKCθ expression leads to aberrant cell proliferation, migration and invasion resulting in malignant phenotype. The recent development and application of PKCθ inhibitors in the context of autoimmune diseases could benefit the emergence of treatment for cancers in which PKCθ has been implicated.

Hepatopulmonary syndrome delays postoperative recovery and increases pulmonary complications after hepatectomy.

BACKGROUND: This study attempted to investigate the impact of hepatopulmonary syndrome (HPS) on postoperative outcomes in hepatitis B virus-induced hepatocellular carcinoma (HBV-HCC) patients. METHODS: HBV-HCC patients undergoing primary curative hepatectomy for HCC in our hospital were diagnosed with HPS by contrast-enhanced echocardiography (CEE) and arterial blood gas analysis. Patients were divided into HPS, intrapulmonary vascular dilation (IPVD) (patients with positive CEE results and normal oxygenation) and control (patients with negative CEE results) groups. Baseline information, perioperative clinical data and postoperative pulmonary complications (PPCs) were compared among all groups. Cytokines in patient serums from each group (n = 8) were also assessed. RESULTS: Eighty-seven patients undergoing hepatectomy from October 2019 to January 2020 were analyzed. The average time in the postanaesthesia care unit (112.10 ± 38.57 min) and oxygen absorption after extubation [34.0 (14.5-54.5) min] in the HPS group was longer than in IPVD [81.81 ± 26.18 min and 16.0 (12.3-24.0) min] and control [93.70 ± 34.06 min and 20.5 (13.8-37.0) min] groups. There were no significant differences in oxygen absorption time after extubation between HPS and control groups. The incidence of PPCs, especially bi-lateral pleural effusions in the HPS group (61.9%), was higher than in IPVD (12.5%) and control (30.0%) groups. Increased serum levels of the growth-regulated oncogene, monocyte chemoattractant protein, soluble CD40 ligand and interleukin 8 might be related to delayed recovery in HPS patients. CONCLUSIONS: HPS patients with HBV-HCC suffer delayed postoperative recovery and are at higher risk for PPCs, especially bi-lateral pleural effusions, which might be associated with changes in certain cytokines.

PKCtheta promotes c-Rel-driven mammary tumorigenesis in mice and humans by repressing estrogen receptor alpha synthesis.

The vast majority of primary human breast cancer tissues display aberrant nuclear NF-kappaB c-Rel expression. A causal role for c-Rel in mammary tumorigenesis has been demonstrated using a c-Rel transgenic mouse model; however, tumors developed with a long latency, suggesting a second event is needed to trigger tumorigenesis. Here we show that c-Rel activity in the mammary gland is repressed by estrogen receptor alpha (ERalpha) signaling, and we identify an epigenetic mechanism in breast cancer mediated by activation of what we believe is a novel PKCtheta-Akt pathway that leads to downregulation of ERalpha synthesis and derepression of c-Rel. ERalpha levels were lower in c-Rel-induced mammary tumors compared with normal mammary gland tissue. PKCtheta induced c-Rel activity and target gene expression and promoted growth of c-Rel- and c-RelxCK2alpha-driven mouse mammary tumor-derived cell lines. RNA expression levels of PKCtheta and c-Rel target genes were inversely correlated with ERalpha levels in human breast cancer specimens. PKCtheta activated Akt, thereby inactivating forkhead box O protein 3a (FOXO3a) and leading to decreased synthesis of its target genes, ERalpha and p27(Kip1). Thus we have shown that activation of PKCtheta inhibits the FOXO3a/ERalpha/p27(Kip1) axis that normally maintains an epithelial cell phenotype and induces c-Rel target genes, thereby promoting proliferation, survival, and more invasive breast cancer.

A Cdc42-mediated supracellular network drives polarized forces and Drosophila egg chamber extension.

Actomyosin supracellular networks emerge during development and tissue repair. These cytoskeletal structures are able to generate large scale forces that can extensively remodel epithelia driving tissue buckling, closure and extension. How supracellular networks emerge, are controlled and mechanically work still remain elusive. During Drosophila oogenesis, the egg chamber elongates along the anterior-posterior axis. Here we show that a dorsal-ventral polarized supracellular F-actin network, running around the egg chamber on the basal side of follicle cells, emerges from polarized intercellular filopodia that radiate from basal stress fibers and extend penetrating neighboring cell cortexes. Filopodia can be mechanosensitive and function as cell-cell anchoring sites. The small GTPase Cdc42 governs the formation and distribution of intercellular filopodia and stress fibers in follicle cells. Finally, our study shows that a Cdc42-dependent supracellular cytoskeletal network provides a scaffold integrating local oscillatory actomyosin contractions at the tissue scale to drive global polarized forces and tissue elongation.

Activation of adenosine A3 receptor reduces early brain injury by alleviating neuroinflammation after subarachnoid hemorrhage in elderly rats.

The incidence of subarachnoid hemorrhage (SAH) and hazard ratio of death increase with age. Overactivation of microglia contributes to brain damage. This study aimed to investigate the effects of A3 adenosine receptors (A3R) activation on neurofunction and microglial phenotype polarization in the context of SAH in aged rats. The A3R agonist (CI-IB-MECA) and antagonist (MRS1523) were used in the SAH model. Microglia were cultured to mimic SAH in the presence or absence of CI-IB-MECA and/or siRNA for A3R. The neurofunction and status of the microglial phenotype were evaluated. The P38 inhibitor SB202190 and the STAT6 inhibitor AS1517499 were used to explore the signaling pathway. The results showed that SAH induced microglia to polarize to the M(LPS) phenotype both in vivo and in vitro. CI-IB-MECA distinctly skewed microglia towards the M(IL-4) phenotype and ameliorated neurological dysfunction, along with the downregulation of inflammatory cytokines. Knockdown of A3R or inhibition of P38 and/or STAT6 weakened the effects of CI-IB-MECA on microglial phenotypic shifting. Collectively, our findings suggest that activation of A3R exerted anti-inflammatory and neuroprotective effects by regulating microglial phenotype polarization through P38/STAT6 pathway and indicated that A3R agonists may be a promising therapeutic options for the treatment of brain injury after SAH.

Activation of FOXO3a by the green tea polyphenol epigallocatechin-3-gallate induces estrogen receptor alpha expression reversing invasive phenotype of breast cancer cells.

Previously, we showed that the bioactive green tea polyphenol epigallocatechin-3-gallate (EGCG) inhibits growth in soft agar of breast cancer cells with Her-2/neu overexpression. Using gene expression profiling, here we show that EGCG treatment of Her-2/neu-driven mammary tumor cells alters the expression of key regulators in the epithelial to mesenchymal transition (EMT) pathway, reducing invasive phenotype. Specifically, the epithelial genes E-cadherin, gamma-catenin, MTA3, and estrogen receptor alpha (ERalpha) were up-regulated by EGCG, whereas the proinvasive snail gene was down-regulated. Consistently, EGCG inhibited branching colony growth and invasion in Matrigel. EGCG treatment similarly inhibited invasive phenotype of mouse mammary tumor cells driven by Nuclear Factor-kappaB c-Rel and protein kinase CK2, frequently found overexpressed in human breast disease. Recently, we identified the Forkhead box O transcription factor FOXO3a as a major transcriptional regulator of ERalpha. Given the pivotal role of ERalpha in preventing EMT, we hypothesized that the activation of FOXO3a by EGCG plays an important role in the observed reversal of invasive phenotype in ERalpha-positive breast cancer cells. EGCG treatment activated FOXO3a. Ectopic expression of a constitutively active FOXO3a overrode transforming growth factor-beta1-mediated invasive phenotype and induced a more epithelial phenotype, which was dependent on ERalpha expression and signaling. Conversely, a dominant negative FOXO3a reduced epithelial phenotype of ERalpha-low breast cancer cells. These results identify, for the first time, a role for FOXO3a in the inhibition of invasive phenotype in breast cancer cells with active ERalpha signaling and elucidate a novel mechanism whereby EGCG represses EMT of breast cancer cells.

Cyclooxygenase-2 regulates HPS patient serum induced-directional collective HPMVEC migration via PKC/Rac signaling pathway.

Hepatopulmonary syndrome (HPS) is a serious complication in patients with advanced liver disease. The pathological pulmonary angiogenesis contributes to the progression of HPS. Importantly, directional collective migration of endothelial cells is a critical event for pathological angiogenesis. Previously, we have demonstrated that the over-expression of Cyclooxygenase-2 (COX-2) was an important factor in the experimental HPS. However, the role of COX-2 in the directional collective migration of human pulmonary microvascular endothelial cells (HPMVECs) is unclear. Our study aims to evaluate the potential effect of COX-2 in the directional collective migration of HPMVECs under the stimulation of HPS patient serum. In this study, 9 patients with stable liver cirrhosis were screened for presence of HPS. We confirmed that HPS patient serum dramatically promoted the directional collective migration and angiogenesis of HPMVECs, while the COX-2 selective antagonist parecoxib significantly inhibited the directional collective migration of HPMVEC under the stimulation of HPS patient serum. In addition, HPS patient serum significantly upregulated the phosphorylation of PKC and promoted the activation of Rac via COX-2/PGE2 signaling pathway. Notably, silencing PKC activation attenuated the directional collective migration of HPMVEC induced by HPS patient serum. In conclusion, these results indicate that PKC/Rac signaling induced by COX-2 modulates collective directional migration of HPMVEC during pathological pulmonary angiogenesis in HPS patients.

Hepatocyte-derived exosomal MiR-194 activates PMVECs and promotes angiogenesis in hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) is a serious vascular complication in the setting of liver disease. Factors produced by the liver are essential to regulate pulmonary angiogenesis in the pathogenesis of HPS; however, the pathogenic mechanisms of pulmonary angiogenesis are not fully understood. We investigated the role of HPS rat serum exosomes (HEs) and sham-operated rat serum exosomes (SEs) in the regulation of angiogenesis. We found that HEs significantly enhance PMVEC proliferation, migration, and tube formation. We further identified miR-194 was the most notably increased miRNA in HEs compared to SEs. Once released, hepatocyte-derived exosomal miR-194 was internalized by PMVECs, leading to the promotion of PMVEC proliferation, migration, and tube formation through direct targeting of THBS1, STAT1, and LIF. Importantly, the pathogenic role of exosomal miR-194 in initiating angiogenesis was reversed by P53 inhibition, exosome secretion inhibition or miR-194 inhibition. Additionally, high levels of miR-194 were found in serum exosomes and were positively correlated with P(A-a)O2 in HPS patients and rats. Thus, our results highlight that the exosome/miR-194 axis plays a critical pathologic role in pulmonary angiogenesis, representing a new therapeutic target for HPS.

MiR145-5p inhibits proliferation of PMVECs via PAI-1 in experimental hepatopulmonary syndrome rat pulmonary microvascular hyperplasia.

Hepatopulmonary syndrome (HPS) is a triad of advanced liver disease, intrapulmonary vasodilatation and arterial hypoxemia. Increasing evidence shows that HPS is associated with pulmonary microvascular hyperplasia. The aim of this work was to investigate the underlying mechanism of miR-145 in regulating the proliferation of pulmonary microvascular endothelial cells (PMVECs) and angiogenesis in HPS via plasminogen activator inhibitor-1 (PAI-1). To test this, morphology score and number of pulmonary microvascular were assessed in lung tissues from rats with HPS by Hematoxylin and Eosin (H&E) staining. Expression levels of PAI-1 were assessed in lung tissues from HPS rats, as well as in PMVECs treated with HPS rat serum. We also selected the putative microRNA binding site on PAI-1 by bioinformatics analysis. Then, miR145-3p and miR145-5p expression levels in the lungs and PMVECs of rats were detected by qRT-PCR because miR145-5p is a microRNA binding site on PAI-1. In addition, the effects of miR-145-5p regulation on PAI-1 were examined by upregulation and downregulation of miR-145-5p and specific lentivirus transfection was used to overexpress and knockdown PAI-1 to assess PAI-1 function on PMVECs proliferation. Our data showed that levels of PAI-1 expression in lung tissue of rats increased significantly when rats were treated with common bile duct ligation. We found that levels of miR-145-5p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-145-5p dramatically inhibited PMVECs proliferation. We further verified PAI-1 as a novel and direct target of miR-145-5p in HPS. MiR-145-5p inhibits PAI-1 synthesis and the expression changes of PAI-1 directly affect the proliferation of PMVECs. We concluded that miR-145-5p negatively regulates PMVEC proliferation through PAI-1 expression. In addition, overexpression of miR-145-5p may prove beneficial as a therapeutic strategy for HPS treatment.

Cyclooxygenase-2 promotes pulmonary intravascular macrophage accumulation by exacerbating BMP signaling in rat experimental hepatopulmonary syndrome.

BACKGROUND AND AIMS: One central factor in hepatopulmonary syndrome (HPS) pathogenesis is intravascular accumulation of activated macrophages in small pulmonary arteries. However, molecular mechanism underlying the macrophage accumulation in HPS is unknown. In this study, we aimed to explore whether elevated COX-2 induces the Bone morphogenic protein-2 (BMP-2)/Crossveinless-2 (CV-2) imbalance and then activation of BMP signaling pathway promotes the macrophage accumulation in Common Bile Duct Ligation (CBDL) rat lung. METHODS: The COX-2/PGE2 signaling activation, the BMP-2/CV-2 imbalance and the activation of Smad1 were evaluated in CBDL rat lung and in cultured pulmonary microvascular endothelial cells (PMVECs) under the HPS serum stimulation. The effects of Parecoxib (COX-2 inhibitor), BMP-2 and CV-2 recombinant proteins on 4-week CBDL rat lung were determined, respectively. RESULTS: The COX-2/PGE2 signaling pathway was activated in CBDL rat lung in vivo and PMVECs in vitro, which was due to the activation of NF-κB P65. The inhibition of COX-2 by Parecoxib reduced macrophage accumulation, decreased lung angiogenesis and improved HPS. Meanwhile, the CBDL rat lung secreted more BMP-2 but less CV-2, and the imbalance between BMP-2 and CV-2 exacerbated the BMP signaling activation thus promoting the macrophage accumulation and lung angiogenesis. The BMP-2/CV-2 imbalance is dependent on the COX-2/PGE2 signaling pathway, and thus the effects of this imbalance can be reversed by adminstration of Parecoxib. CONCLUSION: Our findings indicate that inhibition of COX-2 by parecoxib can improve the HPS through the repression of BMP signaling and macrophage accumulation.