Tumors were harvested and stored at −80°C for subsequent tests. The details of the yeast two-hybrid analysis are in the Supporting Materials. All data were evaluated using SPSS v. 13.5. Gefitinib solubility dmso Differences were considered significant at P < 0.05. The significant groups are marked with an asterisk in the figures. Bcl-2 is an important mitochondrial membrane pore component

that functions in a variety of proapoptotic stress responses, such as hypoxia. In the present study the growth response and hypoxia-induced up-regulation of Bcl-2 in the hepatoma cell lines HepG2, PLC, and SMMC7221, as well as in control cells, were examined. To prevent hypoxia-induced cell death and general protein degradation caused by energy depletion, each cell type was returned to normal oxygen conditions (hypoxia-normoxia group, H-N) after 24 hours of hypoxia. Each cell line showed a significant decrease in cell proliferation following hypoxia, which was reversed by normoxia conditions (H-N) to proliferation levels above control values (Fig. 1A). Notably, the proliferation rate at the terminal phase (72 hours) of the H-N group significantly increased compared Cabozantinib concentration with the normoxia alone control group. Migration and invasion assays showed similar responses (Fig. 1A). Cell migration and invasion

decreased following hypoxia. In contrast, the H-N treatment caused an increase above the control group. HepG2 cultures were also assessed for their abilities to undergo morphological conversion. This conversion leads to VM in a three-dimensional (3D) culture following hypoxia and after returning to normoxia. Cells grown under hypoxic conditions showed a modest level of conversion to “tube” formations, whereas those grown under control conditions did not show any indication of such 3D growth. In contrast, cells that were first treated learn more under hypoxic conditions and were then returned to normoxia showed a robust conversion to 3D tube formations (Fig. 1B). The expression levels of Bcl-2 and Twist following hypoxia and after returning to normoxia were assessed using quantitative PCR and western blot (Fig. 1C,D). Messenger

RNA (mRNA) and protein levels showed expression peaks for Bcl-2 and Twist1 about 24 hours after cell hypoxia. The expression levels gradually decreased to undetectable levels at later timepoints. When hypoxia was relieved after 24 hours by returning to normoxia, Bcl-2 and Twist1 still had high expression levels. Taken together, these observations suggested that return to normoxia after hypoxia may trigger an increase in cell proliferation, movement, and molding. All these functional responses lead to VM, and may be mechanistically linked to the expression levels of Bcl-2 as well as Twist1. The cellular response described above included EMT features. Therefore, EMT markers in HepG2 cells engineered to overexpress Bcl-2 and Twist1, separately or together, were assessed.

7A-C). See the Results section of the Supporting Materials for further details. EGI-1, TFK-1, and CCA1 were selected for experiments with human fibroblasts (Fig. 3 and Supporting Figs. 8 and 9).[8] Effects of conditioned media from CCA Galunisertib cells on fibroblast proliferation (MTS assay) and migration (Boyden chamber) were studied

before and after addition of imatinib, a PDGFRβ antagonist. Effects of EGI-1 cells on fibroblast recruitment were also tested after treatment with siRNA for PDGF-D, resulting in a significant down-regulation of PDGF-D secretion (of approximately 35%-40%, as compared with scramble, P < 0.01 with siRNA1, P < 0.05 with siRNA2) (Supporting Fig. 8). As compared with starved fibroblasts, or with

fibroblasts exposed to conditioned medium Selleck Temozolomide derived from control cholangiocytes, human fibroblasts showed only a mild increase in proliferative activity after exposure to conditioned media from the different CCA cells (from 7% to 15%, as compared to control cholangiocytes) (Fig. 3A). PDGFRβ blockade induced a significant reduction in the rate of proliferating cells in fibroblasts stimulated by EGI-1 and TFK-1 (P < 0.01 and P < 0.05, respectively). As compared to control cholangiocytes, all conditioned media from the different CCA cells induced a potent migration of human fibroblasts (increase of approximately 73%-74%), which reduced significantly after PDGFRβ blockade (P < 0.05 for all CCA cells) (Fig. 3B). Notably, in EGI-1 cells, both PDGF-D siRNA showed a significant reduction in fibroblast recruitment of an extent similar to PDGFRβ blocker (P < 0.05, as compared with scramble). Human fibroblasts exposed to rhPDGF-D

exhibit a similar behavior. Effects of rhPDGF-D on migration were significantly reduced when fibroblasts were exposed to imatinib. These results are detailed in the Supporting Materials and shown in Supporting Fig. 9. To study the signaling pathways activated by PDGFRβ in response to PDGF-D, we stimulated human fibroblasts with rhPDGF-D at increasing doses (0.1, 1, 10, and 100 ng/mL), and then modulation of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated JNK (p-JNK) expression (by western blotting) see more and activation of RhoA, Rac1, and Cdc42 (by G-LISA) were evaluated in the presence or absence of imatinib treatment (Figs. 4 and 5 and Supporting Fig. 10). To determine the kinetics of activation of RhoA, Rac1, and Cdc42, preliminary G-LISA experiments were run at 1, 10, 20, 30, and 60 minutes after stimulation with rhPDGF-D (100 ng/mL). PDGF-D induced a significant increase of p-ERK1/2 only at the highest doses (P < 0.05 at 10 and 100 ng/mL), those able to stimulate also fibroblast proliferation, and this effect was abrogated by imatinib (Fig. 4A). In contrast, increase of p-JNK was significant starting from the lowest doses of rhPDGF-D (0.1 ng/mL; P < 0.01) and was abolished by imatinib (P < 0.01) (Fig. 4B).

7A-C). See the Results section of the Supporting Materials for further details. EGI-1, TFK-1, and CCA1 were selected for experiments with human fibroblasts (Fig. 3 and Supporting Figs. 8 and 9).[8] Effects of conditioned media from CCA find more cells on fibroblast proliferation (MTS assay) and migration (Boyden chamber) were studied

before and after addition of imatinib, a PDGFRβ antagonist. Effects of EGI-1 cells on fibroblast recruitment were also tested after treatment with siRNA for PDGF-D, resulting in a significant down-regulation of PDGF-D secretion (of approximately 35%-40%, as compared with scramble, P < 0.01 with siRNA1, P < 0.05 with siRNA2) (Supporting Fig. 8). As compared with starved fibroblasts, or with

fibroblasts exposed to conditioned medium PXD101 clinical trial derived from control cholangiocytes, human fibroblasts showed only a mild increase in proliferative activity after exposure to conditioned media from the different CCA cells (from 7% to 15%, as compared to control cholangiocytes) (Fig. 3A). PDGFRβ blockade induced a significant reduction in the rate of proliferating cells in fibroblasts stimulated by EGI-1 and TFK-1 (P < 0.01 and P < 0.05, respectively). As compared to control cholangiocytes, all conditioned media from the different CCA cells induced a potent migration of human fibroblasts (increase of approximately 73%-74%), which reduced significantly after PDGFRβ blockade (P < 0.05 for all CCA cells) (Fig. 3B). Notably, in EGI-1 cells, both PDGF-D siRNA showed a significant reduction in fibroblast recruitment of an extent similar to PDGFRβ blocker (P < 0.05, as compared with scramble). Human fibroblasts exposed to rhPDGF-D

exhibit a similar behavior. Effects of rhPDGF-D on migration were significantly reduced when fibroblasts were exposed to imatinib. These results are detailed in the Supporting Materials and shown in Supporting Fig. 9. To study the signaling pathways activated by PDGFRβ in response to PDGF-D, we stimulated human fibroblasts with rhPDGF-D at increasing doses (0.1, 1, 10, and 100 ng/mL), and then modulation of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated JNK (p-JNK) expression (by western blotting) selleck and activation of RhoA, Rac1, and Cdc42 (by G-LISA) were evaluated in the presence or absence of imatinib treatment (Figs. 4 and 5 and Supporting Fig. 10). To determine the kinetics of activation of RhoA, Rac1, and Cdc42, preliminary G-LISA experiments were run at 1, 10, 20, 30, and 60 minutes after stimulation with rhPDGF-D (100 ng/mL). PDGF-D induced a significant increase of p-ERK1/2 only at the highest doses (P < 0.05 at 10 and 100 ng/mL), those able to stimulate also fibroblast proliferation, and this effect was abrogated by imatinib (Fig. 4A). In contrast, increase of p-JNK was significant starting from the lowest doses of rhPDGF-D (0.1 ng/mL; P < 0.01) and was abolished by imatinib (P < 0.01) (Fig. 4B).

It is increasingly recognized that cirrhotic or cholestatic RG7204 order patients show abnormal renal histology with glomerular and tubulointerstitial lesions that may not be noted by routine renal function tests. Microscopic urinanalysis is readily available, inexpensive and noninvasive, and currently considered to be a well-suited surrogate parameter for structural kidney damage. We hypothesized that cirrhotic or cholestatic patients with

preserved renal function (eGFR >60 ml/min) upon routine laboratory evaluation frequently show structural renal injury reflected by a pathologic urine cytology. This may represent a herald of subsequent impaired renal function. Aim: To find a useful non-invasive clinical test to identify early structural kidney injury Acalabrutinib in liver patients. Material and Methods: We collected blood and urine samples from a total of 150 patients [liver cirrhosis Child Pugh score class A (n=41), B (n=38), C (n=28), obstructive cholestasis (n=19), and age-matched healthy living kidney donors (n=24]. Patients with diabetes, insufficiently treated arterial hypertension or preexisting kidney disease were excluded. Freshly voided urine samples were analyzed by automatic flow cytometry (Sysmex UF 1000) and microscopic urinanalysis after Papanicolaou

staining of a smear preparation of the urine sedimentation. The specimens were

analyzed for presence and number of renal tubular epithelial cells (RTEC) and granular casts (GC). Results: Serum creatinine (SCr) concentrations (in mg/dL) and GFR determined by the CKD-EPI equation (in ml/h/1.73m2) were normal amongst all groups (0.76±0.16 selleck chemicals llc and 102±15 in Childs A group, 0.78±0.17 and 101±12 in Childs B group, 0.84±0.23 and 95±19 in Childs C group, 0.85±0.2 and 93±21 in cholestasis group, 0.78±0.11 and 93.6±12.4 in living kidney donors). RTEC and GC as sensitive markers of tubular epithelial kidney injury were frequently found in liver cirrhosis (RTEC in 15%, GC in 8%) and cholestasis (RTEC in 33%, GC in 20%), whereas none of the healthy living kidney donors showed RTEC or GC upon urine cytology. Presence of RTEC significantly correlated with serum bile acid levels (correlation coefficient 0.207; p 0.015) Conclusions: Patients with cirrhosis or cholestasis and normal kidney function show RTEC and GC at increased numbers compared to controls. Microscopic urinanalysis may represent a useful, noninvasive and cheap diagnostic test to identify patients at high risk for AKI or subclinical kidney injury which needs to be evaluated in prospective clinical trials. Disclosures: none.

It is increasingly recognized that cirrhotic or cholestatic PF-01367338 chemical structure patients show abnormal renal histology with glomerular and tubulointerstitial lesions that may not be noted by routine renal function tests. Microscopic urinanalysis is readily available, inexpensive and noninvasive, and currently considered to be a well-suited surrogate parameter for structural kidney damage. We hypothesized that cirrhotic or cholestatic patients with

preserved renal function (eGFR >60 ml/min) upon routine laboratory evaluation frequently show structural renal injury reflected by a pathologic urine cytology. This may represent a herald of subsequent impaired renal function. Aim: To find a useful non-invasive clinical test to identify early structural kidney injury learn more in liver patients. Material and Methods: We collected blood and urine samples from a total of 150 patients [liver cirrhosis Child Pugh score class A (n=41), B (n=38), C (n=28), obstructive cholestasis (n=19), and age-matched healthy living kidney donors (n=24]. Patients with diabetes, insufficiently treated arterial hypertension or preexisting kidney disease were excluded. Freshly voided urine samples were analyzed by automatic flow cytometry (Sysmex UF 1000) and microscopic urinanalysis after Papanicolaou

staining of a smear preparation of the urine sedimentation. The specimens were

analyzed for presence and number of renal tubular epithelial cells (RTEC) and granular casts (GC). Results: Serum creatinine (SCr) concentrations (in mg/dL) and GFR determined by the CKD-EPI equation (in ml/h/1.73m2) were normal amongst all groups (0.76±0.16 selleck screening library and 102±15 in Childs A group, 0.78±0.17 and 101±12 in Childs B group, 0.84±0.23 and 95±19 in Childs C group, 0.85±0.2 and 93±21 in cholestasis group, 0.78±0.11 and 93.6±12.4 in living kidney donors). RTEC and GC as sensitive markers of tubular epithelial kidney injury were frequently found in liver cirrhosis (RTEC in 15%, GC in 8%) and cholestasis (RTEC in 33%, GC in 20%), whereas none of the healthy living kidney donors showed RTEC or GC upon urine cytology. Presence of RTEC significantly correlated with serum bile acid levels (correlation coefficient 0.207; p 0.015) Conclusions: Patients with cirrhosis or cholestasis and normal kidney function show RTEC and GC at increased numbers compared to controls. Microscopic urinanalysis may represent a useful, noninvasive and cheap diagnostic test to identify patients at high risk for AKI or subclinical kidney injury which needs to be evaluated in prospective clinical trials. Disclosures: none.

Host genetic factors are emerging as key elements in the risk for the development of cancer, and the interaction of numerous polymorphisms on a countless genes products, combined with environmental triggers may provide crucial clues explaining diverse risks in various populations. Understanding the molecular mechanisms and alterations behind the initiation and progression of gastric tumorigenesis

is crucial for the early detection of the disease and to identify novel therapeutic and clinical targets for GC. A number of molecular abnormalities have been identified in GC, namely gene overexpression and gene silencing, and MSI-associated gene mutations. Nevertheless, the molecular pathogenesis of GC is still incompletely understood. Over the last decade, a vast amount of articles referring to the overexpression Ivacaftor of various genes in GC was published. Some of those genes were classified as activated oncogenes, like Her-2/neu [26] and c-Myc [27], playing roles in the induction of cell

proliferation. Following the search for other deregulated genes that are involved in cell proliferation, Pan et al. reported the overexpression of SEMA5A (Semaphorin 5A) in GC [28]. With in vitro models, and using siRNA-mediated semaphorin 5A knockdown, those authors concluded that semaphorin 5A may be involved in gastric carcinogenesis by promoting cell proliferation and inhibiting apoptosis. In another study, Selleckchem Deforolimus Florou et al. [29] described how BCL2L12, a member of the BCL2 family that could function as an anti-apoptotic factor, was overexpressed in early stages of GC compared to normal mucosa. The histone-modifying enzymes are responsible for acetylation, phosphorylation, and methylation of histone proteins, playing a key role in the regulation of gene transcription by mediating selleck products chromatin reconfiguration [30]. Zeng et al. [31] described the overexpression of histone demethylase

RBP2 in GC, and they observed that RBP2 depletion triggers the senescence of malignant cells at least partially by derepressing CDKIs. It is known that GC shows a high frequency of DNA aneuploidy [32], and it was recently described that knockdown or overexpression of spindle assembly checkpoint molecules resulted in ploidy errors and carcinogenesis in mice [33]. Knowing that, Ando et al. [34] assessed the expression of BUBR1 kinase, one of the key molecules in the spindle assembly checkpoint, in GC samples. These authors observed a high expression of BUBR1 in GCs that were aneuploid, establishing a relation between BUBR1 expression and induction of aneuploidy. To confirm that association, they enforced expression of BUBR1 in cell lines and, as a result, they observed changes in the ploidy of the cells. Gene silencing in GC can occur mainly because of the point mutations, loss of heterozygosity, and promoter hypermethylation [2,3]. Genetic alterations were reported by Sangodkar et al.

Host genetic factors are emerging as key elements in the risk for the development of cancer, and the interaction of numerous polymorphisms on a countless genes products, combined with environmental triggers may provide crucial clues explaining diverse risks in various populations. Understanding the molecular mechanisms and alterations behind the initiation and progression of gastric tumorigenesis

is crucial for the early detection of the disease and to identify novel therapeutic and clinical targets for GC. A number of molecular abnormalities have been identified in GC, namely gene overexpression and gene silencing, and MSI-associated gene mutations. Nevertheless, the molecular pathogenesis of GC is still incompletely understood. Over the last decade, a vast amount of articles referring to the overexpression BMS-354825 price of various genes in GC was published. Some of those genes were classified as activated oncogenes, like Her-2/neu [26] and c-Myc [27], playing roles in the induction of cell

proliferation. Following the search for other deregulated genes that are involved in cell proliferation, Pan et al. reported the overexpression of SEMA5A (Semaphorin 5A) in GC [28]. With in vitro models, and using siRNA-mediated semaphorin 5A knockdown, those authors concluded that semaphorin 5A may be involved in gastric carcinogenesis by promoting cell proliferation and inhibiting apoptosis. In another study, selleck screening library Florou et al. [29] described how BCL2L12, a member of the BCL2 family that could function as an anti-apoptotic factor, was overexpressed in early stages of GC compared to normal mucosa. The histone-modifying enzymes are responsible for acetylation, phosphorylation, and methylation of histone proteins, playing a key role in the regulation of gene transcription by mediating selleck inhibitor chromatin reconfiguration [30]. Zeng et al. [31] described the overexpression of histone demethylase

RBP2 in GC, and they observed that RBP2 depletion triggers the senescence of malignant cells at least partially by derepressing CDKIs. It is known that GC shows a high frequency of DNA aneuploidy [32], and it was recently described that knockdown or overexpression of spindle assembly checkpoint molecules resulted in ploidy errors and carcinogenesis in mice [33]. Knowing that, Ando et al. [34] assessed the expression of BUBR1 kinase, one of the key molecules in the spindle assembly checkpoint, in GC samples. These authors observed a high expression of BUBR1 in GCs that were aneuploid, establishing a relation between BUBR1 expression and induction of aneuploidy. To confirm that association, they enforced expression of BUBR1 in cell lines and, as a result, they observed changes in the ploidy of the cells. Gene silencing in GC can occur mainly because of the point mutations, loss of heterozygosity, and promoter hypermethylation [2,3]. Genetic alterations were reported by Sangodkar et al.

1, 2 Whereas activated caspase-8 directly activates effector caspases such as caspase-3 and caspase-7 through the so-called extrinsic pathway, leading to apoptosis in type I cells, it activates caspase-3/7 through the mitochondrial pathway in type II cells. In type II cells, activated

caspase-8 cleaves the BH3-only protein Bid into its truncated form, which in turn directly or selleckchem indirectly activates and homo-oligomerizes Bak and/or Bax to form pores at the mitochondrial outer membrane, leading to the release of cytochrome c. After being released, cytochrome c assembles with Apaf-1 to form apoptosomes which promote self-cleavage of procaspase-9 followed by activation of caspase-3/7 to cleave a variety of cellular substrates such as poly(adenosine diphosphate ribose) polymerase (PARP) and finally to

execute apoptosis.8, 9 Hepatocytes are considered to be typical type II cells, because Bid knockout (KO) mice were reported to be resistant to hepatocyte apoptosis upon Fas activation.10, 11 Although Bak and Bax are crucial gateways to apoptosis of the mitochondrial pathway, little information is available about their significance in hepatocyte apoptosis because most traditional Bak/Bax double knockout (DKO) mice (bak−/−bax−/−) die perinatally.12 In the present study, we tried to address this issue by generating hepatocyte-specific Bak/Bax DKO mice. We demonstrate that either Bak or Selleckchem Omipalisib Bax is required and sufficient to induce Fas-mediated early-onset hepatocyte apoptosis and lethal liver injury. Importantly, even if deficient in both Bak and Bax, Bak/Bax DKO mice still develop delayed-onset caspase-dependent massive hepatocyte apoptosis, suggesting that the mitochondria-independent pathway of apoptosis, as observed in type I cells, works as a backup system when the mitochondrial pathway of apoptosis in the liver is absent. This study is the first to demonstrate the significant but limited role of Bak and Bax in

executing Fas-induced apoptosis in the liver. ALT, alanine aminotransferase; find more CypD, cyclophilin D; DISC, death-inducing signaling complex; DKO, double knockout; DMSO, dimethylsulfoxide; IAP, inhibition of apoptosis protein; KO, knockout; PARP, poly(adenosine diphosphate ribose) polymerase; RIP, receptor-interacting protein; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; WT, wild-type. Heterozygous Alb-Cre transgenic mice expressing Cre recombinase gene under the promoter of the albumin gene were described.13 We purchased Bak KO mice (bak−/−), Bax KO mice (bax−/−), and Bak KO mice carrying the bax gene flanked by 2 loxP sites (bak−/−baxflox/flox) from the Jackson Laboratory (Bar Harbor, ME). Traditional cyclophilin D (CypD) KO mice have been described.

[4, 5] Recently, a host genetic

factor, i.e., the DEPDC5 locus polymorphism, was reported to be associated selleck products with progression to HCC in HCV-infected individuals.[6] On the other hand, it remains controversial as to whether HCV itself plays a direct role in the development of HCC. Experimental data suggest that HCV contributes to HCC by modulating pathways that promote malignant transformation of hepatocytes. HCV core, NS3, and NS5A proteins were shown to be involved in a number of potentially oncogenic pathways in cell culture and experimental animal systems.[7] HCV core protein rendered cultured cells more resistant to apoptosis[8, 9] and promoted ras oncogene-mediated transformation.[10, 11] Moreover, transgenic mice expressing the HCV core protein in the liver developed HCC.[12] However, the clinical impact of HCV proteins on HCC development in humans and whether all HCV isolates are equally associated with HCC is yet to be determined. In a clinical setting, HCV core protein mutations at positions 70 (Gln70) and/or 91 (Met91) were closely associated with HCC development.[13] Gln70 and/or Met91 were also linked to resistance Fostamatinib ic50 to PEG-IFN/ribavirin (RBV) treatment.[17] In addition, we and other

investigators reported that an N-terminal part of the NS3 protein has the capacity to transform NIH3T3 and rat fibroblast cells[21, 22] and to render NIH3T3 cells more resistant to DNA damage-induced apoptosis, which is thought to be a prerequisite for malignant transformation of the cell.[23] Also, the NS5A protein is a pleiotropic protein with key roles in both viral learn more RNA replication and modulation of the host cell functions.[24] In particular, the links between NS5A and the IFN responses have been widely discussed. It was proposed initially that sequence variations within a region in NS5A spanning from amino acids (aa) 2209 to 2248, called the IFN sensitivity-determining region (ISDR), were correlated with IFN responsiveness.[25] Subsequently, in the era of PEG-IFN/RBV combination therapy, we identified a new region near the C-terminus of NS5A spanning from

aa 2334 to 2379, which we referred to as the IFN/RBV resistance-determining region (IRRDR).[26, 27] The degree of sequence variations within the IRRDR was significantly associated with the clinical outcome of PEG-IFN/RBV therapy. In the context of HCC, several retrospective studies suggested that IFN-based therapy might reduce the risk of HCC development.[4, 28] In an attempt to clarify whether viral factors, in particular those within the core, NS3, and NS5A proteins, are involved in HCC development, we carried out a comparative analysis of the aa sequences obtained from HCV patients who developed HCC and those who did not. In addition, we studied the sequence evolution of these genes in the interval between chronic hepatitis C and HCC development over a period of 15 years.

More specifically, of the eight cases with SHh+ ballooned hepatocytes, only two showed SHh+ periportal hepatocytes and in these two cases, less than 25% of the portal tracts showed periportal hepatocellular SHh positivity. Conversely, most of the cases with SHh+ periportal hepatocytes

showed no SHh+ ballooned hepatocytes. Of the two cases with SHh+ periportal hepatocytes and SHh+ ballooned hepatocytes, three or fewer SHh+ ballooned hepatocytes were identified per ×100 magnification. On the other hand, SHh+ bile duct/ductular cells tended to be associated with SHh+ periportal hepatocytes, and (like SHh+ periportal hepatocytes) were rarely noted in livers with prevalent SHh+ ballooned hepatocytes. The intensity of SHh+ periportal hepatocellular staining was significantly positively associated with the percentage

of portal BYL719 ic50 tracts showing SHh+ periportal hepatocytes (P < 0.0009) and negatively associated with numbers of SHh+ ballooned hepatocytes (Fig. 3F,G). Gli2+ staining in portal tracts cells was observed in all cases examined (n = 18). The distribution of the grades of Gli2+ portal tract staining was: GDC-0941 mouse G1, 27.8%; G2, 38.9%; and G3, 33.3%. K7+ ductular cells (i.e., liver progenitor cells) were also identified in all cases evaluated (n = 25). The distribution of the grades of K7+ positivity was: G1, 27.8%; G2, 27.8%; and G3, 44.4%. Gli2+ staining and K7+ staining increased with fibrosis stage (Fig. 4A,B). There was a significant positive association between grades of Gli2 portal tract staining and grades of K7 staining (P < 0.017, Fig. 4C). Gli2+ cells were also located in the hepatic lobule in 13 out of the 18 cases,

showing either a zone 3-dominant pattern (Fig. 4D, n = 4), or a zone 1 dominant pattern (Fig. 4E, n = 1) or a combination of zone 1- and zone 3-positivity (n = 8). The pattern of Gli2 staining in the lobule did not show an association with any of the histologic features. In a small number of cases (n = 5), we costained for SHh ligand and the liver progenitor marker, K7. Interesting relationships between SHh positivity and K7 positivity were revealed. learn more All the cases with more than minimal K7 staining (n = 4) showed SHh+ bile duct cells and mild to moderate SHh+ periportal hepatocytes, while the one case with minimal K7 positivity did not show any SHh+ bile duct cells or periportal hepatocytes. The aggregate data, therefore, link portal/periportal production of Hh ligands with accumulation of immature liver cells in the portal/periportal progenitor niche (e.g., ductal plate remnant). Because it is difficult to acquire liver tissue from healthy children to map development-related changes in Hh pathway activity, we performed this analysis in liver sections harvested from healthy male mice at different timepoints during development.