Cholangiocyte-Derived Exosomal Long Noncoding RNA PICALM-AU1 Promotes Pulmonary Endothelial Cell EndMT in Hepatopulmonary Syndrome

Background: Hepatopulmonary syndrome (HPS) is an important clinical problem with limited understanding of disease pathologies. Exosome mediated cell-cell communication can modulate various cellular functions by transferring a variety of intracellular components to target cells. A new lncRNA PICALM-AU1 was found and upregulated in the liver of subjects with HPS. However, the expression and biological functions of the lncRNA PICALM-AU1 are still unknown. Methods: HPS rat model was constructed by common bile duct ligation (CBDL). RNA macroarray was used to analyze the expression differential lncRNAs in HPS rat liver. PICALM-AU1 expression in the serum exosome was measured in 56 HPS patients and in 73 patients with liver cirrhosis but not HPS. qPCR, Fluorescence in situ hybridization were used to analyze PICALM-AU1 expression and location. Virus derived PICALM-AU1 upregulation and down regulation were applied in rats and PMVECs cells. The effects of PICALM-AU1 on PMVECs was determined via CCK8 assay and transwell assay. PICALM-AU1 and miR144-3p relationship was analysis by Dual-luciferase reporter assay.Results: In this study, we found lncRNA PICALM-AU1 expressed in the cholangiocyte of liver, secreted as exosome into the serum. PICALM-AU1 carrying serum exosomes induced endothelial-mesenchymal transition (EndMT) of PMVECs and promoted lung injury. Furthermore, overexpression of PICALM-AU1 significantly suppressed miR144-3p and subsequently induced ZEB1 expression.Conclusions: Taken together, our findings present a road map of targeting the newly identified cholangiocyte-derived exosomal lncRNA PICALM-AU1 plays a critical role in the pathologic angiogenesis of HPS by promoting EndMT and represents a potential therapeutic target for HPS.

Endogenous miR-21 and TIMP-1 Regulate Hepatic Injury and Fibrosis by Bile Duct Ligation in Vivo

TIMP metallopeptidase inhibitor 1 (TIMP-1) has been identified as a multifunctional molecule with divergent functions. It participates in wound healing and regeneration, cell morphology and survival, tumor metastasis, angiogenesis, and inflammatory responses. An imbalance of Matrix Metalloproteinase/TIMP regulation has been implicated in several inflammatory diseases. TIMP-1 could be considered an important regulator in the process of liver fibrosis and bile duct degeneration. Thus, we aimed to determine the role of TIMP-1 in a rat model of Common Bile Duct Ligation (CBDL). Male Sprague-Dawley rats were divided into several groups, including those with/ without CBDL surgery and those with/without amiodarone or simvastatin administration. Amiodarone/simvastatin treatment was given at a daily dose of 15 mg/kg and 18 mg/kg by means of intergalactic gavage, which began 7 days prior to CBDL induction. Two weeks after surgery, the animals in each group were sacrificed and hepatocyte degeneration severity was examined using histological morphologies. Large-scale array for secretory factors is intended for the purpose of finding key functional protein after CBDL. The hepatic level of miR-21 was determined through Taqman miRNA analysis. Furthermore, the TIMP-1 level in liver tissue was also visualized by histological stain. Liver injury and fibrosis were founded in CBDL rats based upon histopathological examination and serum biochemical analysis. Hepatic miR-21 and TIMP-1 were significantly up-regulated in CBDL rats, while being slightly rescued in response to amiodarone or simvastatin treatment. Up-regulation of miR-21 and TIMP-1 may result in the progression of hepatic cirrhosis after bile duct obstruction. Drug intervention for cirrhosis, like the use of statin, may function via similar mechanisms.

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.

Body distribution of stable copper isotopes during the progression of cholestatic liver disease induced by common bile duct ligation in mice

The effect of cholestatic liver disease on the body Cu isotopic distribution was investigated in a common bile duct ligation mouse model. The isotopic composition of Cu in serum and organs becomes gradually lighter with increasing severity of the disease.

Insulin reverses major portal hypertension-related derangements in rats with liver cirrhosis and diabetes

Liver cirrhosis is accompanied by increased intrahepatic resistance and angiogenesis-related portosystemic collaterals formation. Diabetic patients suffer from abnormal vasoresponsiveness and angiogenesis that can be ameliorated by glucose control. However, the relevant presentation is not clear in those with cirrhosis and diabetes, in whom insulin is the treatment of choice. Liver cirrhosis was induced in Sprague–Dawley rats with common bile duct ligation (BDL) and sham rats were used as controls. Streptozotocin 60 mg/kg (STZ, i.p., to induce diabetes) or vehicle was injected. The rats received BDL and STZ injections were injected with insulin or vehicle. On the 29th day after the procedure, the groups were surveyed for (1) systemic and portal hemodynamics; (2) mesenteric vascular density; (3) severity of portosystemic collaterals; (4) hepatic resistance using in situ liver perfusion; (5) histology survey of mesentery and liver; and (6) mesentery angiogenesis- and liver fibrogenesis-related protein expressions. Compared with the cirrhotic rats, the cirrhotic diabetic rats had lower body weight, cardiac output, superior mesenteric arterial (SMA) resistance and portal venous (PV) resistance, and higher SMA and PV flow, which were mostly reversed by insulin. The cirrhotic diabetic rats also had increased mesenteric vascular density, and enhanced pERK, pAkt, VEGF, VEGFR2 protein expressions that were reversed by insulin. Insulin decreased the degree of shunting in the diabetic cirrhotic rats. Hepatic perfusion pressure and severity of liver fibrosis were not significantly influenced by diabetes and insulin treatment in the cirrhotic rats. In conclusion, diabetes aggravated hemodynamic derangements, mesenteric angiogenesis and collaterals in the cirrhotic rats, which were mostly ameliorated by insulin. Further clinical investigations are warranted.

Endotoxemia-enhanced renal vascular reactivity to endothelin-1 in cirrhotic rats

Hepatorenal syndrome (HRS), a severe complication of advanced cirrhosis, is defined as hypoperfusion of kidneys resulting from intense renal vasoconstriction in response to generalized systemic arterial vasodilatation. Nevertheless, the mechanisms have been barely investigated. Cumulative studies demonstrated renal vasodilatation in portal hypertensive and compensated cirrhotic rats. Previously, we identified that blunted renal vascular reactivity of portal hypertensive rats was reversed after lipopolysaccharide (LPS). This study was therefore conducted to delineate the sequence of renal vascular alternation and underlying mechanisms in LPS-treated cirrhotic rats. Sprague-Dawley rats were randomly allocated to receive sham surgery (Sham) or common bile duct ligation (CBDL). LPS was induced on the 28th day after surgery. Kidney perfusion was performed at 0.5 or 3 h after LPS to evaluate renal vascular response to endothelin-1 (ET-1). Endotoxemia increased serum ET-1 levels ( P < 0.0001) and renal arterial blood flow ( P < 0.05) in both Sham and CBDL rats. CBDL rats showed enhanced renal vascular reactivity to ET-1 at 3 h after LPS ( P = 0.026). Pretreatment with endothelin receptor type A (ETA) antagonist abrogated the LPS-enhanced renal vascular response in CBDL rats ( P < 0.001). There were significantly lower inducible nitric oxide synthase (iNOS) expression but higher ETA and phosphorylated extracellular signal-regulated kinase (p-ERK) expressions in renal medulla of endotoxemic CBDL rats ( P < 0.05). We concluded that LPS-induced renal iNOS inhibition, ETA upregulation, and subsequent ERK signaling activation may participate in renal vascular hyperreactivity in cirrhosis. ET-1-targeted therapy may be feasible in the control of HRS. NEW & NOTEWORTHY Hepatorenal syndrome (HRS) occurred in advanced cirrhosis after large-volume paracentesis or bacterial peritonitis. We demonstrated that intraperitoneal lipopolysaccharide (LPS) enhanced renal vascular reactivity to endothelin-1 (ET-1) in cirrhotic rats, accompanied by inducible nitric oxide synthase inhibition, endothelin receptor type A (ETA) upregulation, and subsequent extracellular signal-regulated kinase activation in renal medulla. Pretreatment with ETA antagonist abrogated the LPS-enhanced renal vascular response in common bile duct ligation rats. These findings suggest that further clinical investigation of ET-1-targeted therapy may be feasible in the control of HRS.