Bin Yi

A comparison of two common bile duct ligation methods to establish hepatopulmonary syndrome animal models

The major drawback of the current common bile duct ligation (CBDL)-induced hepatopulmonary syndrome (HPS) animal model is the extremely high mortality rate that hinders experimental studies. The purpose of this study was to investigate an improved method of CBDL with the goal of developing a simple and reproducible rat HPS model after a single CBDL treatment. Two groups of male Sprague–Dawley rats underwent separate methods of CBDL: (1) the upper common bile duct ligation (UCBDL) group (nu2009=u200940), in which the first ligature was made near the junction of the hepatic ducts, and the second ligature was made above the entrance of the pancreatic duct; (2) the middle of the common bile duct ligation (MCBDL) group (nu2009=u200940), in which the first ligature was made in the middle of the common bile duct, and the second ligature was made above the entrance of the pancreatic duct. The CBDL-induced HPS rats were evaluated by pulse oximeter, arterial blood analysis, histopathology, and cerebral uptake of intravenous technetium-99m-labeled albumin macroaggregates (which reflects intrapulmonary vascular dilation). The mortality rates of the UCBDL group and the MCBDL group were 42.5% and 77.5%, respectively (Pu2009<u20090.05). These results suggest that the UCBDL, a single improved procedure, provides a better method compared to the established HPS model, because of the relatively high success rate and the decreased risk of complications.

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.

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.

Erythropoietin ameliorates early brain injury after subarachnoid haemorrhage by modulating microglia polarization via the EPOR/JAK2-STAT3 pathway

ABSTRACT Inflammatory modulation mediated by microglial M1/M2 polarization is one of the main pathophysiological processes involved in early brain injury (EBI) after subarachnoid haemorrhage (SAH). Previous studies have shown that recombinant human erythropoietin (rhEPO) alleviates EBI following experimental SAH. However, the mechanisms of this beneficial effect are still poorly understood. Recent research has suggested that EPO shows anti‐inflammatory properties. Therefore, we tried to analyse whether rhEPO administration influenced microglial M1/M2 polarization in early brain injury after SAH and to identify the underlying molecular mechanism of any such effect. We found that treatment with rhEPO markedly ameliorated SAH‐induced EBI, as shown by reductions in brain cell apoptosis, neuronal necrosis, albumin exudation and brain edema. Moreover, the expression levels of p‐JAK2 and p‐STAT3 were significantly increased in the cortex after SAH induction and were further increased by EPO treatment; in addition, the p‐JAK2 inhibitor AZD1480 impaired the protective effect of EPO against SAH‐induced EBI in vivo. Furthermore, EPO promoted the polarization of microglia towards the protective M2 phenotype and alleviated inflammation. In cultured microglia under oxyhemoglobin (OxyHb) treatment, EPO up‐regulated the expression of the EPO receptor (EPOR), which did not occur in response to OxyHb treatment alone, and EPO magnified OxyHb‐induced increases in p‐JAK2 and p‐STAT3 and modulated OxyHb‐challenged microglial polarization towards M2. Interestingly, the effect of EPO on microglia polarization was cancelled by EPOR knockdown or by p‐JAK2 or p‐STAT3 inhibition, suggesting a core role of the EPOR/JAK2/STAT3 pathway in modulating microglial function and phenotype. In conclusion, the therapeutic effect of rhEPO on the early brain injury after SAH may relate to its modulation of inflammatory response and microglia M1/M2 polarization, which may be mediated in part by the EPOR/JAK2/STAT3 signalling pathway. These results improved the understanding of the anti‐inflammatory effect of EPO on microglia polarization, which might optimize the therapeutic modalities of EPO treatment with SAH. HIGHLIGHTSEPO post‐treatment alleviates early brain injury (EBI) following subarachnoid haemorrhage (SAH).Microglial M2 polarization mitigates SAH‐induced inflammation.EPO modulates microglia polarization towards M2 via the EPOR/JAK2‐STAT3 pathway.

Inhibition of autophagy ameliorates pulmonary microvascular dilation and PMVECs excessive proliferation in rat experimental hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) is a defective liver-induced pulmonary vascular disorder with massive pulmonary microvascular dilation and excessive proliferation of pulmonary microvascular endothelial cells (PMVECs). Growing evidence suggests that autophagy is involved in pulmonary diseases, protectively or detrimentally. Thus, it is interesting and important to explore whether autophagy might be involved in and critical in HPS. In the present study, we report that autophagy was activated in common bile duct ligation (CBDL) rats and cultured pulmonary PMVECs induced by CBDL rat serum, two accepted in vivo and in vitro experimental models of HPS. Furthermore, pharmacological inhibition of autophagy with 3-methyladenine (3-MA) significantly alleviated pathological alterations and typical symptom of HPS in CBDL rats in vivo, and consistently 3-MA significantly attenuated the CBDL rat serum-induced excessive proliferation of PMVECs in vitro. All these changes mediated by 3-MA might explain the observed prominent improvement of pulmonary appearance, edema, microvascular dilatation and arterial oxygenation in vivo. Collectively, these results suggest that autophagy activation may play a critical role in the pathogenesis of HPS, and autophagy inhibition may have a therapeutic potential for this disease.

Hemodynamic Monitoring in Pigs Undergoing Orthotopic Abdominal Multivisceral Transplantation

UNLABELLEDnAnesthesia for abdominal multivisceral transplantation (MVTx) is challenging. Surgical success depends on hemodynamic stability throughout organ removal from the donor and engraftment in recipients. The aim of the current study was to summarize our experience with anesthetic management including monitoring of hemodynamic changes in pigs during MVTx procedures.nnnMATERIALS AND METHODSnTen male pigs were randomly divided into two groups: donors and recipients for five MVTxs. A pulmonary artery catheter (PAC), transesophageal echocardiography (TEE), and pulse indicator continuous cardiac output (PiCCO) were used intraoperatively in recipients to monitor hemodynamic changes.nnnRESULTSnTwo recipients had cardiac arrest and died intraoperatively presumably due to hypovolemia. The data obtained by PAC and TEE from three successful cases showed huge changes in hemodynamics during the stages of organ removal and reperfusion after engraftment. Use of PiCOO in one of those three cases allowed successful fluid management, resulting in stable intraoperative hemodynamics.nnnCONCLUSIONnThe anesthetic management for MVTx surgery was improved by PAC, TEE, and PiCOO monitoring.

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.

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‐&kgr;B 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. HIGHLIGHTSCOX‐2/PGE2 signaling is strongly activated in lung after CBDL.COX‐2/PGE2 activation after CBDL promotes the development of HPS via macrophage accumulation.The BMP‐2/CV‐2 imbalance induced BMP signaling promotes macrophage accumulation after CBDL.COX‐2/PGE2 signaling promotes the imbalance of BMP‐2/CV‐2 in PMVECs and lung.Parecoxib improves HPS by the inhibition of BMP‐2/CV‐2 imbalance and macrophage accumulation, indicating its clinical value.

The angiogenic related functions of bone marrow mesenchymal stem cells are promoted by CBDL rat serum via the Akt/Nrf2 pathway.

Hepatopulmonary syndrome (HPS) is a complication of severe liver disease. It is characterized by an arterial oxygenation defect. Recent studies have demonstrated that pulmonary angiogenesis contributes to the abnormal gas exchange found in HPS. Additionally, mesenchymal stem cells (MSCs) are considered the stable source of VEGF-producing cells and have the potential to differentiate into multiple cell types. However, it has not been determined whether bone marrow mesenchymal stem cells (BM-MSCs) are mobilized and involved in the pulmonary angiogenesis in HPS. In this study, a CFU-F assay showed that the number of peripheral blood MSCs was increased in common bile duct ligation (CBDL) rats; however, there was no significant difference found in the number of BM-MSCs. In vitro, CBDL rat serum induced the overexpression of CXCR4 and PCNA in BM-MSCs. Consistently, the directional migration as well as the proliferation ability of BM-MSCs were enhanced by CBDL rat serum, as determined by a transwell migration and MTT assays. Moreover, the secretion of VEGF by BM-MSCs increased after treatment with CBDL rat serum. We also found that the expression of phospho-Akt, phospho-ERK, and Nrf2 in BM-MSCs was significantly up-regulated by CBDL rat serum in a time dependent manner, and the blockage of the Akt/Nrf2 signalling pathway with an Akt Inhibitor or Nrf2 siRNA, instead of an ERK inhibitor, attenuated the migration, proliferation and paracrine capacity of BM-MSCs. In conclusion, these findings indicated that the number of MSCs increased in the peripheral blood of CBDL rats, and the Akt/Nrf2 pathway plays a vital role in promoting the angiogenic related functions of BM-MSCs, which could be a potent contributor to pulmonary angiogenesis in HPS.

A biochemical network controlling basal myosin oscillation

The actomyosin cytoskeleton, a key stress-producing unit in epithelial cells, oscillates spontaneously in a wide variety of systems. Although much of the signal cascade regulating myosin activity has been characterized, the origin of such oscillatory behavior is still unclear. Here, we show that basal myosin II oscillation in Drosophila ovarian epithelium is not controlled by actomyosin cortical tension, but instead relies on a biochemical oscillator involving ROCK and myosin phosphatase. Key to this oscillation is a diffusive ROCK flow, linking junctional Rho1 to medial actomyosin cortex, and dynamically maintained by a self-activation loop reliant on ROCK kinase activity. In response to the resulting myosin II recruitment, myosin phosphatase is locally enriched and shuts off ROCK and myosin II signals. Coupling Drosophila genetics, live imaging, modeling, and optogenetics, we uncover an intrinsic biochemical oscillator at the core of myosin II regulatory network, shedding light on the spatio-temporal dynamics of force generation.The actomyosin cytoskeleton is known to spontaneously oscillate in many systems but the mechanism of this behavior is not clear. Here Qin et al. define a signaling network involving a ROCK-dependent self-activation loop and recruitment of myosin II to the cortex, followed by a local accumulation of myosin phosphatase that shuts off the signal.