- Email: [email protected]
Tu1698 Modulation of Epithelial Mesenchymal Transition by miRNA125b in Cholestatic Bile Duct Ligated (BDL) Mice
following by stimulation with recombinant FGF. Results: Genetic deletion of Msln attenuated cholestatic fibrosis in Msln-/- mice, and was associated with reduced number of aPFs in livers of BDL-injured Msln-/- mice. In vitro analysis demonstrated that Msln-/- aPFs exhibit a defect in proliferation (compared with wt aPFs). Msln-/- aPFs failed to populate the scratch area within 12h and exhibited a defect in expression of proliferation marker Ki67. Expression of Cyclin D mRNA was suppressed (fl4.5 fold) in FGF-stimulated Msln-/- aPFs, and correlated with downregulation of c-Myc, integrin-b6, and MMP9 mRNA. Furthermore, re-expression of Msln in Msln-/- aPFs (by transfection with Msln-pCMV construct) restored basal and FGF-mediated proliferation of Mslnko+Msln aPFs, as shown by increased numbers of cells populating the scratch area and upregulation of Cyclin D mRNA. Mechanistic studies demonstrated that FGF-mediated proliferation of aPFs is regulated by JAK2-STAT3 and ERK1/2 signaling pathways. Expression of Cyclin D was most effectively (>80%) suppressed in wt and Msln-/- aPFs by JAK2/ERK inhibitor. In concordance, proliferation and migration of wt and Msln-/- aPFs to the scratch area was completely blocked in the presence of JAK2/ERK inhibitor and was associated with complete inhibition of ERK1/2 and STAT3 phosphorylation. Conclusion: Mesothelin regulates proliferation of aPFs via ERK1/2 and JAK2-STAT3 pathways.
pathway components are differentially expressed during HSC activation and loss of RALDH1 abrogates transition of cultured HSCs to a myofibroblast phenotype, suggesting it regulates HSC activation.
Tu1698
AASLD Abstracts
Modulation of Epithelial Mesenchymal Transition by miRNA125b in Cholestatic Bile Duct Ligated (BDL) Mice Maria Cristina Navas, Nan Wu, Julie Venter, Fanyin Meng, Paolo Onori, Eugenio Gaudio, Shannon Glaser, Gianfranco Alpini Cholangiocytes are the target of cholestatic liver diseases such as primary sclerosing cholangitis that are characterized by biliary proliferation/damage and the progression of fibrosis. Biliary damage and liver fibrosis are coordinately regulated by a number of neuroendocrine factors including secretin. We have previously shown that: (i) secretin stimulates biliary growth by downregulation of miR125b; and (ii) miRNA125b is downregulated in cholestatic bile duct ligated (BDL) and mdr2-/- mice. miRNA125b has been shown to play a key role in the regulation of epithelial mesenchymal transition (EMT). Moreover, a number of studies have shown that the progression of liver fibrosis is associated with enhanced EMT. The aim of this study was to evaluate the role of miRNA125b in the modulation of liver fibrosis by secretin through changes in EMT. METHODS: Liver tissue was collected from wild-type (WT) mice underwent sham surgery or BDL for 7 days. The mice were treated by tailvein injections with Vivo Morpholino sequence against miRNA125b (to reduce the hepatic expression of miRNA125b) or mismatched Morpholino at days 3 and 7 after surgery. Studies were also performed in normal WT and mdr2-/- mice treated with secretin by osmotic minipumps (10 ng/kg BW/day) during 7 days. Thereafter, we measured: (i) liver fibrosis by Sirius red staining in liver sections and by qPCR for transforming growth factor-beta1 (TGF-beta1), collagen type 1 (Col1A1) and fibronectin-1 (Fn-1) in total liver tissue; and (ii) EMT by qPCR for N-cadherin and Vimentin in total liver tissue. RESULTS: We observed an increase in the % of connective tissue in liver sections and enhanced expression of TGFbeta1, Col1A1 and Fn-1 in normal and BDL mice treated with Vivo miRNA125b Morpholino, and normal WT and mdr2-/- mice chronically treated with secretin (where miRNA125b levels are decreased) compared to control mice. In BDL mice treated with Vivo miRNA125b Morpholino and normal WT and mdr2-/- mice treated with secretin, there was enhanced expression of N-Cadherin and Vimentin compared to respective control mice. CONCLUSION: We have demonstrated that downregulation of miR125b by secretin induces liver fibrosis by selective upregulation of EMT. Delivery of miRNA125b mimics may be an important therapeutic approach to ameliorate biliary damage and liver fibrosis by modulation of EMT.
Figure 1. Freshly harvested (A) and culture-activated (B) normal rat HSCs have fewer and larger lipid droplets than BDL-injured rat HSCs (C, D) at day 1 (A, C) and day 4 in culture (B, D). After isolation, and culture for the specified time, cells were formalin fixed and stained with oil red o. Using ImageJ, photomicrographs were converted to binary images to better demonstrate lipid size and number for further quantification. Single representative cells are shown for each condition and time point.
Figure 2. Molecular knockdown of RALDH1 in HSCs results in loss of stress fibers and normal cellular adhesion. HSCs harvested from normal rat were cultured for 2 days prior to transduction with siRNA targeting scrambled (A, C) or RALDH1 (B, D). Loss of RALDH1 protein was verified by immunoblotting (data not shown) and resulted in cell rounding and loss of stress fibers (B) when compared to control (A) (a single representative cell is shown at 20x magnification). Lower magnification (10x) shows that knockdown of RALDH1 results in cell clumping (D) compared to control cells (C).
Tu1699 Lipid Droplet Loss and Retinoid Pathway Mediators Differ in Normal and BDL Activated Hepatic Stellate Cells Loretta Jophlin, Don C. Rockey
Tu1700 Background: Hepatic stellate cells (HSCs) undergo "activation," the transition to a myofibroblast phenotype during liver injury and are the primary effectors of liver fibrosis. A core feature of HSC activation is loss of vitamin A-laden lipid droplets (LDs). It is unclear whether LD loss is a causal or secondary event in HSC activation. Retinaldehyde dehydrogenase 1 (RALDH1) is a critical regulator of vitamin A signaling. Given the importance of RALDH1 in vitamin A signaling, and the association of LD loss with fibrosis, we hypothesized that RALDH1 regulates HSC activation. Methods: HSCs from control rats and those with bile duct ligation (BDL)-induced liver injury were isolated and cultured. LDs were assessed using a novel oil red O/microscopic quantification technique to assess LD phenotype and velocity of loss. We measured RALDH1 expression and two other retinoid pathway members, the vitamin A cellular uptake receptor, stimulated by retinoic acid 6 (STRA6), and the enzyme responsible for the storage of vitamin A derivatives into LDs, lecithin retinol acyltransferase (LRAT) with real-time PCR, immunoblotting, and immunocytochemistry. Finally, we examined the cellular effects of knockdown of these three retinoid pathway components. Results: In freshly-isolated HSCs, normal quiescent HSCs had fewer LDs than in vivo activated BDL HSCs (5.3±1.6 vs 10.8±6.7 droplets/cell, n>8, p=0.004). Lipid droplets were larger in normal HSCs than BDL HSCs (311±114 vs 98.3±89 pixels/cell, n>8, p=0.006, Figure 1). Over time in culture, total LD number increased as average droplet size decreased in both normal and injured HSCs. LDs were absent at day14 in normal HSCs and day 9 in BDL HSCs. As such, the velocity of LD loss over time was more rapid in BDL-HSCs than in culture-activated HSCs (8.6 vs 3.4 pixels/hour). We found that RALDH1, STRA6 and LRAT mRNA expression was increased in both normal and BDL cultured HSCs (RALDH, 3-fold; STRA6, 2-fold; and LRAT, 2-fold). Each retinoid pathway mediator had a unique subcellular localization pattern that changed over the course of HSC activation; most prominently, RALDH1 localized to stress fibers in activated HSCs. Finally, RALDH1 knockdown in quiescent HSCs downregulated smooth muscle alpha actin and beta-actin, both major constituents of the cytoskeleton. RALDH1 knockdown also had functional effects on HSCs, causing abnormalities in lamellipodia, stress fibers, cell size and cell adhesion ( Figure 2). Conclusions: The number, size and velocity of LD loss over time are different in normal and BDL-injured HSCs. Retinoid
AASLD Abstracts
X : 10052$CH02 03-28-16 22:36:37 PDFd : 10052B : e
Radiomics of Computed Tomography (CT) Liver Scans: A Novel Method to Evaluate Liver Cirrhosis and Steatosis Neil B. Marya, Suman Shrestha, Srinivasan Vedantham, Krunal Patel, Abbas H. Rupawala, Summer Aldrugh, Adib R. Karam, Isabel Zacharias, Andrew Karellas Background: CT scan of the liver is an important imaging tool in the diagnosis of hepatic cirrhosis and steatosis. Current interpretation is based on morphological visual analysis and the difference in Hounsfield Units (HU) between the liver and the spleen. Radiomics is the extraction of descriptors of tissue radiomic features that allows identification of patterns that differ with histopathology and facilitates objective quantitative analysis. We hypothesize that radiomics of CT images of the liver and the spleen can differentiate between normal and abnormal livers. Methods: In this IRB-approved retrospective study, records of patients diagnosed as having liver disease were screened and included if found to have a diagnosis of either steatosis or cirrhosis based on histologic, imaging, and laboratory evidence. CT urograms of patients with no prior history of liver disease and with no CT appearance of abnormal morphology served as normal livers. Only non-contrast CT studies of the liver were included. Thirty eight radiomic features each of the liver and spleen based on 1st and 2nd order statistics were extracted and along with reconstructed CT slice thickness and HU difference between liver and spleen (HU-Diff) served as potential predictors. Dimensionality reduction was achieved by correlation analysis, variable clustering, collinearity analysis and the data was modeled using multivariable logistic regression. All logistic regression models employed leave one out cross-validation for ROC analysis. Results: A total of 112 cases were included in the study (46 normal, 20 steatosis and 46 cirrhosis). After adjusting for CT slice thickness, the area under the ROC (AUC [95% CI]) for discriminating between normal and abnormal (steatosis or cirrhosis) livers and between normal and cirrhotic livers were 0.923 [0.876,0.971] and 0.886 [0.821,0.952], respectively, with HU-Diff (p<0.008) and a Fourier based radiomic feature (p<0.003) as significant predictors. For discriminating between normal and steatosis livers the AUC was 0.971 [0.939, 1.0] with HU-Diff (p<0.001)
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pathway components are differentially expressed during HSC activation and loss of RALDH1 abrogates transition of cultured HSCs to a myofibroblast phenotype, suggesting it regulates HSC activation.
Tu1698
AASLD Abstracts
Modulation of Epithelial Mesenchymal Transition by miRNA125b in Cholestatic Bile Duct Ligated (BDL) Mice Maria Cristina Navas, Nan Wu, Julie Venter, Fanyin Meng, Paolo Onori, Eugenio Gaudio, Shannon Glaser, Gianfranco Alpini Cholangiocytes are the target of cholestatic liver diseases such as primary sclerosing cholangitis that are characterized by biliary proliferation/damage and the progression of fibrosis. Biliary damage and liver fibrosis are coordinately regulated by a number of neuroendocrine factors including secretin. We have previously shown that: (i) secretin stimulates biliary growth by downregulation of miR125b; and (ii) miRNA125b is downregulated in cholestatic bile duct ligated (BDL) and mdr2-/- mice. miRNA125b has been shown to play a key role in the regulation of epithelial mesenchymal transition (EMT). Moreover, a number of studies have shown that the progression of liver fibrosis is associated with enhanced EMT. The aim of this study was to evaluate the role of miRNA125b in the modulation of liver fibrosis by secretin through changes in EMT. METHODS: Liver tissue was collected from wild-type (WT) mice underwent sham surgery or BDL for 7 days. The mice were treated by tailvein injections with Vivo Morpholino sequence against miRNA125b (to reduce the hepatic expression of miRNA125b) or mismatched Morpholino at days 3 and 7 after surgery. Studies were also performed in normal WT and mdr2-/- mice treated with secretin by osmotic minipumps (10 ng/kg BW/day) during 7 days. Thereafter, we measured: (i) liver fibrosis by Sirius red staining in liver sections and by qPCR for transforming growth factor-beta1 (TGF-beta1), collagen type 1 (Col1A1) and fibronectin-1 (Fn-1) in total liver tissue; and (ii) EMT by qPCR for N-cadherin and Vimentin in total liver tissue. RESULTS: We observed an increase in the % of connective tissue in liver sections and enhanced expression of TGFbeta1, Col1A1 and Fn-1 in normal and BDL mice treated with Vivo miRNA125b Morpholino, and normal WT and mdr2-/- mice chronically treated with secretin (where miRNA125b levels are decreased) compared to control mice. In BDL mice treated with Vivo miRNA125b Morpholino and normal WT and mdr2-/- mice treated with secretin, there was enhanced expression of N-Cadherin and Vimentin compared to respective control mice. CONCLUSION: We have demonstrated that downregulation of miR125b by secretin induces liver fibrosis by selective upregulation of EMT. Delivery of miRNA125b mimics may be an important therapeutic approach to ameliorate biliary damage and liver fibrosis by modulation of EMT.
Figure 1. Freshly harvested (A) and culture-activated (B) normal rat HSCs have fewer and larger lipid droplets than BDL-injured rat HSCs (C, D) at day 1 (A, C) and day 4 in culture (B, D). After isolation, and culture for the specified time, cells were formalin fixed and stained with oil red o. Using ImageJ, photomicrographs were converted to binary images to better demonstrate lipid size and number for further quantification. Single representative cells are shown for each condition and time point.
Figure 2. Molecular knockdown of RALDH1 in HSCs results in loss of stress fibers and normal cellular adhesion. HSCs harvested from normal rat were cultured for 2 days prior to transduction with siRNA targeting scrambled (A, C) or RALDH1 (B, D). Loss of RALDH1 protein was verified by immunoblotting (data not shown) and resulted in cell rounding and loss of stress fibers (B) when compared to control (A) (a single representative cell is shown at 20x magnification). Lower magnification (10x) shows that knockdown of RALDH1 results in cell clumping (D) compared to control cells (C).
Tu1699 Lipid Droplet Loss and Retinoid Pathway Mediators Differ in Normal and BDL Activated Hepatic Stellate Cells Loretta Jophlin, Don C. Rockey
Tu1700 Background: Hepatic stellate cells (HSCs) undergo "activation," the transition to a myofibroblast phenotype during liver injury and are the primary effectors of liver fibrosis. A core feature of HSC activation is loss of vitamin A-laden lipid droplets (LDs). It is unclear whether LD loss is a causal or secondary event in HSC activation. Retinaldehyde dehydrogenase 1 (RALDH1) is a critical regulator of vitamin A signaling. Given the importance of RALDH1 in vitamin A signaling, and the association of LD loss with fibrosis, we hypothesized that RALDH1 regulates HSC activation. Methods: HSCs from control rats and those with bile duct ligation (BDL)-induced liver injury were isolated and cultured. LDs were assessed using a novel oil red O/microscopic quantification technique to assess LD phenotype and velocity of loss. We measured RALDH1 expression and two other retinoid pathway members, the vitamin A cellular uptake receptor, stimulated by retinoic acid 6 (STRA6), and the enzyme responsible for the storage of vitamin A derivatives into LDs, lecithin retinol acyltransferase (LRAT) with real-time PCR, immunoblotting, and immunocytochemistry. Finally, we examined the cellular effects of knockdown of these three retinoid pathway components. Results: In freshly-isolated HSCs, normal quiescent HSCs had fewer LDs than in vivo activated BDL HSCs (5.3±1.6 vs 10.8±6.7 droplets/cell, n>8, p=0.004). Lipid droplets were larger in normal HSCs than BDL HSCs (311±114 vs 98.3±89 pixels/cell, n>8, p=0.006, Figure 1). Over time in culture, total LD number increased as average droplet size decreased in both normal and injured HSCs. LDs were absent at day14 in normal HSCs and day 9 in BDL HSCs. As such, the velocity of LD loss over time was more rapid in BDL-HSCs than in culture-activated HSCs (8.6 vs 3.4 pixels/hour). We found that RALDH1, STRA6 and LRAT mRNA expression was increased in both normal and BDL cultured HSCs (RALDH, 3-fold; STRA6, 2-fold; and LRAT, 2-fold). Each retinoid pathway mediator had a unique subcellular localization pattern that changed over the course of HSC activation; most prominently, RALDH1 localized to stress fibers in activated HSCs. Finally, RALDH1 knockdown in quiescent HSCs downregulated smooth muscle alpha actin and beta-actin, both major constituents of the cytoskeleton. RALDH1 knockdown also had functional effects on HSCs, causing abnormalities in lamellipodia, stress fibers, cell size and cell adhesion ( Figure 2). Conclusions: The number, size and velocity of LD loss over time are different in normal and BDL-injured HSCs. Retinoid
AASLD Abstracts
X : 10052$CH02 03-28-16 22:36:37 PDFd : 10052B : e
Radiomics of Computed Tomography (CT) Liver Scans: A Novel Method to Evaluate Liver Cirrhosis and Steatosis Neil B. Marya, Suman Shrestha, Srinivasan Vedantham, Krunal Patel, Abbas H. Rupawala, Summer Aldrugh, Adib R. Karam, Isabel Zacharias, Andrew Karellas Background: CT scan of the liver is an important imaging tool in the diagnosis of hepatic cirrhosis and steatosis. Current interpretation is based on morphological visual analysis and the difference in Hounsfield Units (HU) between the liver and the spleen. Radiomics is the extraction of descriptors of tissue radiomic features that allows identification of patterns that differ with histopathology and facilitates objective quantitative analysis. We hypothesize that radiomics of CT images of the liver and the spleen can differentiate between normal and abnormal livers. Methods: In this IRB-approved retrospective study, records of patients diagnosed as having liver disease were screened and included if found to have a diagnosis of either steatosis or cirrhosis based on histologic, imaging, and laboratory evidence. CT urograms of patients with no prior history of liver disease and with no CT appearance of abnormal morphology served as normal livers. Only non-contrast CT studies of the liver were included. Thirty eight radiomic features each of the liver and spleen based on 1st and 2nd order statistics were extracted and along with reconstructed CT slice thickness and HU difference between liver and spleen (HU-Diff) served as potential predictors. Dimensionality reduction was achieved by correlation analysis, variable clustering, collinearity analysis and the data was modeled using multivariable logistic regression. All logistic regression models employed leave one out cross-validation for ROC analysis. Results: A total of 112 cases were included in the study (46 normal, 20 steatosis and 46 cirrhosis). After adjusting for CT slice thickness, the area under the ROC (AUC [95% CI]) for discriminating between normal and abnormal (steatosis or cirrhosis) livers and between normal and cirrhotic livers were 0.923 [0.876,0.971] and 0.886 [0.821,0.952], respectively, with HU-Diff (p<0.008) and a Fourier based radiomic feature (p<0.003) as significant predictors. For discriminating between normal and steatosis livers the AUC was 0.971 [0.939, 1.0] with HU-Diff (p<0.001)
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