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PROMININ-1-expressing Progenitor Cells undergo Transforming Growth Factor-B (TGFB)-mediated Transdifferentiation into Myofibroblasts during Biliary Fibrosis
PEDIATRIC SURGERY I depends on an adequate store of Zn transported into SGs through the transporter ZnT2.
PROMININ-1-expressing Progenitor Cells undergo Transforming Growth Factor-B (TGFB)-mediated Transdifferentiation into Myofibroblasts during Biliary Fibrosis Marie V Nguyen, MD*, Nirmala Mavila, PhD, David James, Kasper S Wang, MD, FACS Children’s Hospital Los Angeles, Los Angeles, CA
METHODS: The following studies were performed using wild-type (WT) and ZnT2-null (KO) mice: 1) immunohistochemistry (IHC) of tissue sections; 2) subcellular fractionation using antibodies to ZnT2 and Reg-III-g in isolated crypts; 3) fluorescence microscopy of Zn accumulation using the fluorescent reporter ZP-1 that visualizes zinc in SGs; and 4) degranulation analysis of lysozyme release after flush of ileal segments with saline.
INTRODUCTION: Biliary atresia (BA), a lethal congenital cholangiopathy, is associated with intrahepatic ductular proliferation within regions of biliary fibrosis. We previously reported the expansion of dual epithelial-mesenchymal cells expressing the stem/progenitor cell marker, PROMININ-1 (PROM1), in a murine model of BA induced by perinatal Rhesus rotavirus (RRV) infection and in human BA. These PROM1+ cells transdifferentiate into Collagen-1a1 producing myofibroblasts during BA-associated fibrogenesis in association with TGFB signaling activation. We hypothesize that PROM1+ derived myofibroblasts promote fibrosis associated with biliary-specific injuries.
RESULTS: Histologically, WT and KO mice were similar (crypt depth, villous height/width, cellularity). However, PCs in KO mice had significantly more degranulated PCs (p<0.01). Localization of ZnT2 in SGs was confirmed both in tissue sections and cofractionation with Reg-III-g. In KO mice, absence of ZnT2 was confirmed by IHC and failure of SGs to take up ZP-1. In response to saline flush, which should not trigger degranulation, ileal segments from KO mice responded with unstimulated release of lysozyme into the lumen (p¼0.03).
METHODS: Biliary fibrosis was induced by RRV, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, and bile duct ligation (BDL). Hepatocyte-induced injury was induced by 70% partial hepatectomy (PH) and carbon tetrachloride (CCl4) treatment. Livers were analyzed histologically for PROM1+ cell expansion and fibrosis. Immortalized PROM1+ progenitor cells derived from Mat1a-/- livers were cultured recombinant(r) TGFB.
CONCLUSIONS: ZnT2 imports zinc into PC granules and plays a significant role in their response to luminal challenges. Individuals with defects in ZnT2 may be at increased risk for enteric infections and inflammatory diseases. Slit3 Knockout Mice with Congenital Diaphragmatic Hernia Develop Pulmonary Arterial Hypertension and Abnormal Vascular Remodeling Michael R Phillips, MD, Jin Li, BS, Clara H Lee, Yueh Z Lee, MD, PhD, JS Marron, PhD, Sean E McLean, MD, FACS University of North Carolina School of Medicine, Chapel Hill, NC
RESULTS: PROM1+ cells were observed in RRV, DDC, and BDL but not in PH or CCl4. Grossly, up to 5 weeks after BDL, livers exhibited evolving gallbladder distention, extrahepatic ductal dilatation, and ascites. PROM1+ cells emerged adjacent to expanding periportal ductular reactions at 3 and 4 weeks after BDL, similar to that of RRV-induced BA. Over 5 weeks, there was a progressive increase in periportal collagen deposition up to 18-fold compared to sham (p<0.001). Cultured PROM1+ progenitor cells undergo morphologic myofibroblastic transdifferentiation with an 80-fold increase in Collagen-1a1 expression with rTGFB treatment (p<0.0001).
INTRODUCTION: The Slit3 knockout (KO) mouse reliably produces a congenital diaphragmatic hernia (CDH) phenotype, and is associated with right ventricular (RV) hypertension and pulmonary arterial hypertension (PAH). PAH occurs due to abnormal pulmonary vascular development and pulmonary vascular remodeling due to unknown mechanisms.
CONCLUSIONS: Emergence of PROM1+ progenitor cells is a precursor to TGFB-mediated myofibroblastic transdifferentiation, which contributes to biliary fibrosis.
METHODS: Slit3 KO mice were bred at our institution. At 3 months of age, wild type (WT) and KO mice were harvested. RV pressures were measured by cardiac puncture. The main pulmonary artery was cannulated and lungs were perfused with Microfil casting agent. MicroCT (Scanco) scans were obtained with 8 mm resolution. CT images of each vessel segment were analyzed using Amira software, vascular tree analysis was performed. Vessel analysis was performed based on size and degree of vessel branching (rank) from the main pulmonary artery.
Zinc Transporter ZnT2 Regulates Activity of Secretory Granules in the Intestinal Paneth Cell Abigail B Podany, MD, Abby K Geletzke, MD, Sooyeon Lee, David I Soybel, MD, FACS, Shannon L Kelleher, PhD Penn State Hershey Medical Center, Hershey, PA INTRODUCTION: Recent studies suggest that Paneth cells (PCs) play a critical role in development of the intestine and self-protection against luminal pathogens. Defects in PC function are associated with Crohn’s disease and necrotizing enterocolitis (NEC). PC secretory granules (SGs) release antimicrobial peptides including lysozyme and Reg-III-g and, remarkably, a high content of zinc (Zn). Here we explore the hypothesis that responsiveness of PCs
ª 2014 by the American College of Surgeons Published by Elsevier Inc.
RESULTS: When compared to Slit3 WT mice, KO mice with CDH had significantly elevated RV systolic (17.2 vs 35.2) and mean (8.2 vs 18.7) pressures, p<0.001. KO mice had a trend toward fewer total vessels per mouse by vessel size. KO mice had significantly increased mean vessel length, mean vessel radius,
S72
http://dx.doi.org/10.1016/j.jamcollsurg.2014.07.169 ISSN 1072-7515/14
PROMININ-1-expressing Progenitor Cells undergo Transforming Growth Factor-B (TGFB)-mediated Transdifferentiation into Myofibroblasts during Biliary Fibrosis Marie V Nguyen, MD*, Nirmala Mavila, PhD, David James, Kasper S Wang, MD, FACS Children’s Hospital Los Angeles, Los Angeles, CA
METHODS: The following studies were performed using wild-type (WT) and ZnT2-null (KO) mice: 1) immunohistochemistry (IHC) of tissue sections; 2) subcellular fractionation using antibodies to ZnT2 and Reg-III-g in isolated crypts; 3) fluorescence microscopy of Zn accumulation using the fluorescent reporter ZP-1 that visualizes zinc in SGs; and 4) degranulation analysis of lysozyme release after flush of ileal segments with saline.
INTRODUCTION: Biliary atresia (BA), a lethal congenital cholangiopathy, is associated with intrahepatic ductular proliferation within regions of biliary fibrosis. We previously reported the expansion of dual epithelial-mesenchymal cells expressing the stem/progenitor cell marker, PROMININ-1 (PROM1), in a murine model of BA induced by perinatal Rhesus rotavirus (RRV) infection and in human BA. These PROM1+ cells transdifferentiate into Collagen-1a1 producing myofibroblasts during BA-associated fibrogenesis in association with TGFB signaling activation. We hypothesize that PROM1+ derived myofibroblasts promote fibrosis associated with biliary-specific injuries.
RESULTS: Histologically, WT and KO mice were similar (crypt depth, villous height/width, cellularity). However, PCs in KO mice had significantly more degranulated PCs (p<0.01). Localization of ZnT2 in SGs was confirmed both in tissue sections and cofractionation with Reg-III-g. In KO mice, absence of ZnT2 was confirmed by IHC and failure of SGs to take up ZP-1. In response to saline flush, which should not trigger degranulation, ileal segments from KO mice responded with unstimulated release of lysozyme into the lumen (p¼0.03).
METHODS: Biliary fibrosis was induced by RRV, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, and bile duct ligation (BDL). Hepatocyte-induced injury was induced by 70% partial hepatectomy (PH) and carbon tetrachloride (CCl4) treatment. Livers were analyzed histologically for PROM1+ cell expansion and fibrosis. Immortalized PROM1+ progenitor cells derived from Mat1a-/- livers were cultured recombinant(r) TGFB.
CONCLUSIONS: ZnT2 imports zinc into PC granules and plays a significant role in their response to luminal challenges. Individuals with defects in ZnT2 may be at increased risk for enteric infections and inflammatory diseases. Slit3 Knockout Mice with Congenital Diaphragmatic Hernia Develop Pulmonary Arterial Hypertension and Abnormal Vascular Remodeling Michael R Phillips, MD, Jin Li, BS, Clara H Lee, Yueh Z Lee, MD, PhD, JS Marron, PhD, Sean E McLean, MD, FACS University of North Carolina School of Medicine, Chapel Hill, NC
RESULTS: PROM1+ cells were observed in RRV, DDC, and BDL but not in PH or CCl4. Grossly, up to 5 weeks after BDL, livers exhibited evolving gallbladder distention, extrahepatic ductal dilatation, and ascites. PROM1+ cells emerged adjacent to expanding periportal ductular reactions at 3 and 4 weeks after BDL, similar to that of RRV-induced BA. Over 5 weeks, there was a progressive increase in periportal collagen deposition up to 18-fold compared to sham (p<0.001). Cultured PROM1+ progenitor cells undergo morphologic myofibroblastic transdifferentiation with an 80-fold increase in Collagen-1a1 expression with rTGFB treatment (p<0.0001).
INTRODUCTION: The Slit3 knockout (KO) mouse reliably produces a congenital diaphragmatic hernia (CDH) phenotype, and is associated with right ventricular (RV) hypertension and pulmonary arterial hypertension (PAH). PAH occurs due to abnormal pulmonary vascular development and pulmonary vascular remodeling due to unknown mechanisms.
CONCLUSIONS: Emergence of PROM1+ progenitor cells is a precursor to TGFB-mediated myofibroblastic transdifferentiation, which contributes to biliary fibrosis.
METHODS: Slit3 KO mice were bred at our institution. At 3 months of age, wild type (WT) and KO mice were harvested. RV pressures were measured by cardiac puncture. The main pulmonary artery was cannulated and lungs were perfused with Microfil casting agent. MicroCT (Scanco) scans were obtained with 8 mm resolution. CT images of each vessel segment were analyzed using Amira software, vascular tree analysis was performed. Vessel analysis was performed based on size and degree of vessel branching (rank) from the main pulmonary artery.
Zinc Transporter ZnT2 Regulates Activity of Secretory Granules in the Intestinal Paneth Cell Abigail B Podany, MD, Abby K Geletzke, MD, Sooyeon Lee, David I Soybel, MD, FACS, Shannon L Kelleher, PhD Penn State Hershey Medical Center, Hershey, PA INTRODUCTION: Recent studies suggest that Paneth cells (PCs) play a critical role in development of the intestine and self-protection against luminal pathogens. Defects in PC function are associated with Crohn’s disease and necrotizing enterocolitis (NEC). PC secretory granules (SGs) release antimicrobial peptides including lysozyme and Reg-III-g and, remarkably, a high content of zinc (Zn). Here we explore the hypothesis that responsiveness of PCs
ª 2014 by the American College of Surgeons Published by Elsevier Inc.
RESULTS: When compared to Slit3 WT mice, KO mice with CDH had significantly elevated RV systolic (17.2 vs 35.2) and mean (8.2 vs 18.7) pressures, p<0.001. KO mice had a trend toward fewer total vessels per mouse by vessel size. KO mice had significantly increased mean vessel length, mean vessel radius,
S72
http://dx.doi.org/10.1016/j.jamcollsurg.2014.07.169 ISSN 1072-7515/14