Comparative Investigation of Anti-Inflammatory Effect of Platelet-Rich Fibrin after Mandibular Wisdom Tooth Surgery: A Randomized Controlled Study.

This study evaluated the anti-inflammatory effect of platelet-rich fibrin (PRF) applied to the extraction socket after impacted mandibular third molar surgery with subjective and objective parameters. Forty-eight patients with impacted wisdom teeth in bilateral and similar positions were included in the study. The control group was formed with the standard surgery and the PRF group was formed with local PRF application in addition to standard procedure (n = 96). The anti-inflammatory activity of PRF on postoperative 2nd and 7th days was evaluated subjectively by clinical parameters and objectively by biochemical parameters. Postoperative 2nd- and 7th-day follow-up data of pain, edema, and trismus in the PRF group were found to be statistically significantly lower. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were found to be statistically significantly lower in the PRF group than the control in the postoperative 2nd-day follow-up period (p < 0.001). There was no statistically significant difference in interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) parameters when the PRF group and the control group were compared in both follow-up periods (p > 0.05). The study has demonstrated the effectiveness of locally applied PRF after ITM surgery via clinical parameters and objective data. The quantitative analysis of CRP and ERS can be an effective parameter in determining the amount of inflammation after ITM surgery.

Does Saline Irrigation at Different Temperatures Affect Pain, Edema, and Trismus After Impacted Third Molar Surgery: A Clinical Trial.

PURPOSE: Lower impacted third molar surgery is a very common oral-maxillofacial surgical procedure, which has complications such as facial swelling, pain, and trismus. This clinical trial aimed to compare the intensity of postoperative morbidity (pain, facial swelling, and trismus) following the third molar surgery performed using saline irrigation at different temperatures (4 °C, 10 °C, or 25 °C). MATERIALS AND METHODS: This double-blind, single-center, split-mouth, randomized prospective clinical trial was conducted among 48 systemically and periodontally healthy patients who had bilaterally asymptomatic mandibular third molars. Patients were randomly allocated into 2 groups (n = 24) according to the temperature of the saline used. In each patient, one impacted third molar was determined as the test group (4 °C or 10 °C saline irrigation) and the other impacted third molar as the control group (25 °C saline irrigation). Trismus and swelling were evaluated on the 1st, 3rd, and 7th days postoperatively. Pain perception by visual analog scale (VAS) and the total number of analgesics taken during the 7 postoperative days were recorded. Data were analyzed using the Shapiro-Wilk test, the chi-square test, one-way analysis of variance, Duncan test, the Kruskal-Wallis test, the Dunn test, and the Friedman test (P < .05). RESULTS: Forty-eight patients (28 females, 20 males) with a mean age of 24.6 ± 3.8 years were included in the study. The duration of operations was similar. VAS values of test groups [test group 1 (4 °C): 4.0, test group 1 (10 °C): 8.0] and the number of analgesics taken [test group 1 (4 °C): 0, test group 1 (10°) C): 3] were significantly lower (P < .001) than control groups (VAS, control group 1: 13.0, control group 2: 15.5, number of analgesic taken, control group 1: 5.5, control group 2: 4.0). Significant differences were found between the test groups in VAS values and the number of analgesics taken (P < .001). Also, the lowest trismus and facial swelling values were detected in the 4 °C test group at all time points (P < .001). CONCLUSION: In the impacted third molar surgery, the use of cooled saline irrigation during bone removal may be a simple, inexpensive, and effective method for reducing early postoperative complaints.

The relationship between caspase-1 related inflammasome expression and serum inflammatory cytokine levels during acute brucellosis.

OBJECTIVE: Brucellosis is a zoonotic disease caused by Brucella in domestic and wild animals. It also causes systemic diseases with the involvement of different parts of the human body. An efficient innate immune response is crucial to cure brucellosis with optimum antibiotic treatment. The inflammasomes are innate immune system receptors and sensors that regulate the activation of cysteine-dependent aspartate specific protease-1 (caspase-1) and caspase-1-induced cell death process known as pyroptosis. The aim of the present study was to investigate the expression levels of CASPASE-1 and associated inflammasomes AIM2, NLRP3, and NLRC4 to analyze their relationship with the inflammatory cytokine interleukin (IL)-1ß, IL-18, and interferon-gamma (IFN-γ) in peripheral blood samples of patients with acute brucellosis with healthy controls. METHODS: Peripheral blood samples were obtained from 20 healthy volunteers and 20 patients with acute brucellosis. RNA and serum samples were isolated to examine the expression levels of AIM2, NLRP3, NLRC4, and CASPASE-1 by real-time polymerase chain reaction, and IL-1ß, IL-18, and IFN-γ were measured by enzyme-linked immunosorbent assay. RESULTS: In the acute brucellosis group, AIM2 and NLRC4 expressions were significantly higher than in healthy volunteers. A significant increase on caspase-1 expression in patients with acute brucellosis was not observed. Serum IL-18 and IFN-γ levels were significantly higher in patients with acute brucellosis than in healthy controls. CONCLUSION: Caspase-1-related inflammasomes are sufficiently activated to induce the secretion of cytokines, such as IFN-γ and IL-18, to induce cellular immune response. Caspase-1 activation level should be investigated at different periods of disease in a group with high number of patients to understand the role of pyroptosis and caspase-1 in brucellosis.

Interleukin-1ß Activates a MYC-Dependent Metabolic Switch in Kidney Stromal Cells Necessary for Progressive Tubulointerstitial Fibrosis.

Background Kidney injury is characterized by persisting inflammation and fibrosis, yet mechanisms by which inflammatory signals drive fibrogenesis remain poorly defined.Methods RNA sequencing of fibrotic kidneys from patients with CKD identified a metabolic gene signature comprising loss of mitochondrial and oxidative phosphorylation gene expression with a concomitant increase in regulators and enzymes of glycolysis under the control of PGC1α and MYC transcription factors, respectively. We modeled this metabolic switch in vivo, in experimental murine models of kidney injury, and in vitro in human kidney stromal cells (SCs) and human kidney organoids.Results In mice, MYC and the target genes thereof became activated in resident SCs early after kidney injury, suggesting that acute innate immune signals regulate this transcriptional switch. In vitro, stimulation of purified human kidney SCs and human kidney organoids with IL-1ß recapitulated the molecular events observed in vivo, inducing functional metabolic derangement characterized by increased MYC-dependent glycolysis, the latter proving necessary to drive proliferation and matrix production. MYC interacted directly with sequestosome 1/p62, which is involved in proteasomal degradation, and modulation of p62 expression caused inverse effects on MYC expression. IL-1ß stimulated autophagy flux, causing degradation of p62 and accumulation of MYC. Inhibition of the IL-1R signal transducer kinase IRAK4 in vivo or inhibition of MYC in vivo as well as in human kidney organoids in vitro abrogated fibrosis and reduced tubular injury.Conclusions Our findings define a connection between IL-1ß and metabolic switch in fibrosis initiation and progression and highlight IL-1ß and MYC as potential therapeutic targets in tubulointerstitial diseases.

TWEAK-Fn14 Signaling Activates Myofibroblasts to Drive Progression of Fibrotic Kidney Disease.

The identification of the cellular origins of myofibroblasts has led to the discovery of novel pathways that potentially drive myofibroblast perpetuation in disease. Here, we further investigated the role of innate immune signaling pathways in this process. In mice, renal injury-induced activation of pericytes, which are myofibroblast precursors attached to endothelial cells, led to upregulated expression of TNF receptor superfamily member 12a, also known as fibroblast growth factor-inducible 14 (Fn14), by these cells. In live rat kidney slices, administration of the Fn14 ligand, TNF-related weak inducer of apoptosis (TWEAK), promoted pericyte-dependent vasoconstriction followed by pericyte detachment from capillaries. In vitro, administration of TWEAK activated and differentiated pericytes into cytokine-producing myofibroblasts, and further activated established myofibroblasts in a manner requiring canonical and noncanonical NF-κB signaling pathways. Deficiency of Fn14 protected mouse kidneys from fibrogenesis, inflammation, and associated vascular instability after in vivo injury, and was associated with loss of NF-κB signaling. In a genetic model of spontaneous CKD, therapeutic delivery of anti-TWEAK blocking antibodies attenuated disease progression, preserved organ function, and increased survival. These results identify the TWEAK-Fn14 signaling pathway as an important factor in myofibroblast perpetuation, fibrogenesis, and chronic disease progression.

Role of Fn14 in acute alcoholic steatohepatitis in mice.

TNF-like weak inducer of apoptosis (TWEAK) is a growth factor for bipotent liver progenitors that express its receptor, fibroblast growth factor-inducible 14 (Fn14), a TNF receptor superfamily member. Accumulation of Fn14(+) progenitors occurs in severe acute alcoholic steatohepatitis (ASH) and correlates with acute mortality. In patients with severe ASH, inhibition of TNF-α increases acute mortality. The aim of this study was to determine whether deletion of Fn14 improves the outcome of liver injury in alcohol-consuming mice. Wild-type (WT) and Fn14 knockout (KO) mice were fed control high-fat Lieber deCarli diet or high-fat Lieber deCarli diet with 2% alcohol (ETOH) and injected intraperitoneally with CCl4 for 2 wk to induce liver injury. Mice were euthanized 3 or 10 days after CCl4 treatment. Survival was assessed. Liver tissues were analyzed for cell death, inflammation, proliferation, progenitor accumulation, and fibrosis by quantitative RT-PCR, immunoblot, hydroxyproline content, and quantitative immunohistochemistry. During liver injury, Fn14 expression, apoptosis, inflammation, hepatocyte replication, progenitor and myofibroblast accumulation, and fibrosis increased in WT mice fed either diet. Mice fed either diet expressed similar TWEAK/Fn14 levels, but ETOH-fed mice had higher TNF-α expression. The ETOH-fed group developed more apoptosis, inflammation, fibrosis, and regenerative responses. Fn14 deletion did not reduce hepatic TNF-α expression but improved all injury parameters in mice fed the control diet. In ETOH-fed mice, Fn14 deletion inhibited TNF-α induction and increased acute mortality, despite improvement in liver injury. Fn14 mediates wound-healing responses that are necessary to survive acute liver injury during alcohol exposure.

TWEAK/Fn14 signaling is required for liver regeneration after partial hepatectomy in mice.

BACKGROUND & AIMS: Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH. METHODS: To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies. RESULTS: In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12-8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes. CONCLUSIONS: TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.

Alcohol activates the hedgehog pathway and induces related procarcinogenic processes in the alcohol-preferring rat model of hepatocarcinogenesis.

BACKGROUND: Alcohol consumption promotes hepatocellular carcinoma (HCC). The responsible mechanisms are not well understood. Hepatocarcinogenesis increases with age and is enhanced by factors that impose a demand for liver regeneration. Because alcohol is hepatotoxic, habitual alcohol ingestion evokes a recurrent demand for hepatic regeneration. The alcohol-preferring (P) rat model mimics the level of alcohol consumption by humans who habitually abuse alcohol. Previously, we showed that habitual heavy alcohol ingestion amplified age-related hepatocarcinogenesis in P rats, with over 80% of alcohol-consuming P rats developing HCCs after 18 months of alcohol exposure, compared with only 5% of water-drinking controls. METHODS: Herein, we used quantitative real-time PCR and quantitative immunocytochemistry to compare liver tissues from alcohol-consuming P rats and water-fed P rat controls after 6, 12, or 18 months of drinking. We aimed to identify potential mechanisms that might underlie the differences in liver cancer formation and hypothesized that chronic alcohol ingestion would activate Hedgehog (HH), a regenerative signaling pathway that is overactivated in HCC. RESULTS: Chronic alcohol ingestion amplified age-related degenerative changes in hepatocytes, but did not cause appreciable liver inflammation or fibrosis even after 18 months of heavy drinking. HH signaling was also enhanced by alcohol exposure, as evidenced by increased levels of mRNAs encoding HH ligands, HH-regulated transcription factors, and HH target genes. Immunocytochemistry confirmed increased alcohol-related accumulation of HH ligand-producing cells and HH-responsive target cells. HH-related regenerative responses were also induced in alcohol-exposed rats. Three of these processes (i.e., deregulated progenitor expansion, the reverse Warburg effect, and epithelial-to-mesenchymal transitions) are known to promote cancer growth in other tissues. CONCLUSIONS: Alcohol-related changes in Hedgehog signaling and resultant deregulation of liver cell replacement might promote hepatocarcinogenesis.

Hedgehog signalling regulates liver sinusoidal endothelial cell capillarisation.

OBJECTIVE: Vascular remodelling during liver damage involves loss of healthy liver sinusoidal endothelial cell (LSEC) phenotype via capillarisation. Hedgehog (Hh) signalling regulates vascular development and increases during liver injury. This study therefore examined its role in capillarisation. DESIGN: Primary LSEC were cultured for 5 days to induce capillarisation. Pharmacological, antibody-mediated and genetic approaches were used to manipulate Hh signalling. Effects on mRNA and protein expression of Hh-regulated genes and capillarisation markers were evaluated by quantitative reverse transcription PCR and immunoblot. Changes in LSEC function were assessed by migration and tube forming assay, and gain/loss of fenestrae was examined by electron microscopy. Mice with acute or chronic liver injury were treated with Hh inhibitors; effects on capillarisation were assessed by immunohistochemistry. RESULTS: Freshly isolated LSEC expressed Hh ligands, Hh receptors and Hh ligand antagonist Hhip. Capillarisation was accompanied by repression of Hhip and increased expression of Hh-regulated genes. Treatment with Hh agonist further induced expression of Hh ligands and Hh-regulated genes, and upregulated capillarisation-associated genes; whereas Hh signalling antagonist or Hh ligand neutralising antibody each repressed expression of Hh target genes and capillarisation markers. LSEC isolated from Smo(loxP/loxP) transgenic mice that had been infected with adenovirus expressing Cre-recombinase to delete Smoothened showed over 75% knockdown of Smoothened. During culture, Smoothened-deficient LSEC had inhibited Hh signalling, less induction of capillarisation-associated genes and retention of fenestrae. In mice with injured livers, inhibiting Hh signalling prevented capillarisation. CONCLUSIONS: LSEC produce and respond to Hh ligands, and use Hh signalling to regulate complex phenotypic changes that occur during capillarisation.

Paracrine Hedgehog signaling drives metabolic changes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) typically develops in cirrhosis, a condition characterized by Hedgehog (Hh) pathway activation and accumulation of Hh-responsive myofibroblasts. Although Hh signaling generally regulates stromal-epithelial interactions that support epithelial viability, the role of Hh-dependent myofibroblasts in hepatocarcinogenesis is unknown. Here, we used human HCC samples, a mouse HCC model, and hepatoma cell/myofibroblast cocultures to examine the hypothesis that Hh signaling modulates myofibroblasts' metabolism to generate fuels for neighboring malignant hepatocytes. The results identify a novel paracrine mechanism whereby malignant hepatocytes produce Hh ligands to stimulate glycolysis in neighboring myofibroblasts, resulting in release of myofibroblast-derived lactate that the malignant hepatocytes use as an energy source. This discovery reveals new diagnostic and therapeutic targets that might be exploited to improve the outcomes of cirrhotic patients with HCCs.

Hedgehog controls hepatic stellate cell fate by regulating metabolism.

BACKGROUND & AIMS: The pathogenesis of cirrhosis, a disabling outcome of defective liver repair, involves deregulated accumulation of myofibroblasts derived from quiescent hepatic stellate cells (HSCs), but the mechanisms that control transdifferentiation of HSCs are poorly understood. We investigated whether the Hedgehog (Hh) pathway controls the fate of HSCs by regulating metabolism. METHODS: Microarray, quantitative polymerase chain reaction, and immunoblot analyses were used to identify metabolic genes that were differentially expressed in quiescent vs myofibroblast HSCs. Glycolysis and lactate production were disrupted in HSCs to determine if metabolism influenced transdifferentiation. Hh signaling and hypoxia-inducible factor 1α (HIF1α) activity were altered to identify factors that alter glycolytic activity. Changes in expression of genes that regulate glycolysis were quantified and localized in biopsy samples from patients with cirrhosis and liver samples from mice following administration of CCl(4) or bile duct ligation. Mice were given systemic inhibitors of Hh to determine if they affect glycolytic activity of the hepatic stroma; Hh signaling was also conditionally disrupted in myofibroblasts to determine the effects of glycolytic activity. RESULTS: Transdifferentiation of cultured, quiescent HSCs into myofibroblasts induced glycolysis and caused lactate accumulation. Increased expression of genes that regulate glycolysis required Hh signaling and involved induction of HIF1α. Inhibitors of Hh signaling, HIF1α, glycolysis, or lactate accumulation converted myofibroblasts to quiescent HSCs. In diseased livers of animals and patients, numbers of glycolytic stromal cells were associated with the severity of fibrosis. Conditional disruption of Hh signaling in myofibroblasts reduced numbers of glycolytic myofibroblasts and liver fibrosis in mice; similar effects were observed following administration of pharmacologic inhibitors of Hh. CONCLUSIONS: Hedgehog signaling controls the fate of HSCs by regulating metabolism. These findings might be applied to diagnosis and treatment of patients with cirrhosis.

Pancreatic secretory trypsin inhibitor I reduces the severity of chronic pancreatitis in mice overexpressing interleukin-1ß in the pancreas.

IL-1ß is believed to play a pathogenic role in the development of pancreatitis. Expression of human IL-1ß in pancreatic acinar cells produces chronic pancreatitis, characterized by extensive intrapancreatic inflammation, atrophy, and fibrosis. To determine if activation of trypsinogen is important in the pathogenesis of chronic pancreatitis in this model, we crossed IL-1ß transgenic [Tg(IL1ß)] mice with mice expressing a trypsin inhibitor that is normally produced in rat pancreatic acinar cells [pancreatic secretory trypsin inhibitor (PTSI) I]. We previously demonstrated that transgenic expression of PSTI-I [Tg(Psti1)] increased pancreatic trypsin inhibitor activity by 190%. Tg(IL1ß) mice were found to have marked pancreatic inflammation, characterized by histological changes, including acinar cell loss, inflammatory cell infiltration, and fibrosis, as well as elevated myeloperoxidase activity and elevated pancreatic trypsin activity, as early as 6 wk of age. In contrast to Tg(IL1ß) mice, pancreatitis was significantly less severe in dual-transgenic [Tg(IL1ß)-Tg(Psti1)] mice expressing IL-1ß and PSTI-I in pancreatic acinar cells. These findings indicate that overexpression of PSTI-I reduces the severity of pancreatitis and that pancreatic trypsin activity contributes to the pathogenesis of an inflammatory model of chronic pancreatitis.

Hedgehog signaling antagonist promotes regression of both liver fibrosis and hepatocellular carcinoma in a murine model of primary liver cancer.

OBJECTIVE: Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50-60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2(-/-) mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC. METHODS: Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2(-/-) mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging. RESULTS: Unlike controls, Mdr2(-/-) mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality. CONCLUSIONS: Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.

Leptin promotes the myofibroblastic phenotype in hepatic stellate cells by activating the hedgehog pathway.

Trans-differentiation of quiescent hepatic stellate cells (Q-HSCs), which exhibit epithelial and adipocytic features, into myofibroblastic-HSC (MF-HSCs) is a key event in liver fibrosis. Culture models demonstrated that Hedgehog (Hh) pathway activation is required for transition of epithelioid/adipocytic Q-HSCs into MF-HSCs. Hh signaling inhibits adiposity and promotes epithelial-to-mesenchymal transitions (EMTs). Leptin (anti-adipogenic, pro-EMT factor) promotes HSC trans-differentiation and liver fibrosis, suggesting that the pathways may interact to modulate cell fate. This study aimed to determine whether leptin activates Hh signaling and whether this is required for the fibrogenic effects of leptin. Cultures of primary HSCs from lean and fa/fa rats with an inherited ObRb defect were examined. Inhibitors of PI3K/Akt, JAK/STAT, and Hh signaling were used to delineate how ObRb activation influenced Hh signaling and HSC trans-differentiation. Fibrogenesis was compared in wild type and db/db mice (impaired ObRb function) to assess the profibrotic role of leptin. The results demonstrate that leptin-ObR interactions activate Hh signaling with the latter necessary to promote trans-differentiation. Leptin-related increases in Hh signaling required ObR induction of PI3K/Akt, which was sufficient for leptin to repress the epithelioid/adipocytic program. Leptin-mediated induction of JAK/STAT was required for mesenchymal gene expression. Leptin-ObRb interactions were not necessary for HSC trans-differentiation to occur in vitro or in vivo but are important because liver fibrogenesis was attenuated in db/db mice. These findings reveal that leptin activates Hh signaling to alter gene expression programs that control cell fate and have important implications for liver fibrosis and other leptin-regulated processes involving EMTs, including development, obesity, and cancer metastasis.

Viral factors induce Hedgehog pathway activation in humans with viral hepatitis, cirrhosis, and hepatocellular carcinoma.

Hedgehog (Hh) pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCCs) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCCs develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarciongenesis in chronic viral hepatitis. Immunohistochemistry and qRT-PCR analysis were used to analyze Hh pathway activation and identify Hh-responsive cell types in liver biopsies from 45 patients with chronic HBV or HCV. Hh signaling was then manipulated in cultured liver cells to directly assess the impact of Hh activity in relevant cell types. We found increased hepatic expression of Hh ligands in all patients with chronic viral hepatitis, and demonstrated that infection with HCV stimulated cultured hepatocytes to produce Hh ligands. The major cell populations that expanded during cirrhosis and HCC (ie, liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells) were Hh responsive, and higher levels of Hh pathway activity associated with cirrhosis and HCC. Inhibiting pathway activity in Hh-responsive target cells reduced fibrogenesis, angiogenesis, and growth. In conclusion, HBV/HCV infection increases hepatocyte production of Hh ligands and expands the types of Hh-responsive cells that promote liver fibrosis and cancer.

Activation of Rac1 promotes hedgehog-mediated acquisition of the myofibroblastic phenotype in rat and human hepatic stellate cells.

UNLABELLED: Hepatic accumulation of myofibroblastic hepatic stellate cells (MF-HSCs) is pivotal in the pathogenesis of cirrhosis. Two events are necessary for MF-HSCs to accumulate in damaged livers: transition of resident, quiescent hepatic stellate cells (Q-HSCs) to MF-HSCs and expansion of MF-HSC numbers through increased proliferation and/or reduced apoptosis. In this study, we identified two novel mediators of MF-HSC accumulation: Ras-related C3 botulinum toxin substrate 1 (Rac1) and Hedgehog (Hh). It is unclear whether Rac1 and Hh interact to regulate the accumulation of MF-HSCs. We evaluated the hypothesis that Rac1 promotes activation of the Hh pathway, thereby stimulating signals that promote transition of Q-HSCs into MF-HSCs and enhance the viability of MF-HSCs. Using both in vitro and in vivo model systems, Rac1 activity was manipulated through adenoviral vector-mediated delivery of constitutively active or dominant-negative rac1. Rac1-transgenic mice with targeted myofibroblast expression of a mutated human rac1 transgene that produces constitutively active Rac1 were also examined. Results in all models demonstrated that activating Rac1 in HSC enhanced Hh signaling, promoted acquisition/maintenance of the MF-HSC phenotype, increased MF-HSC viability, and exacerbated fibrogenesis. Conversely, inhibiting Rac1 with dominant-negative rac1 reversed these effects in all systems examined. Pharmacologic manipulation of Hh signaling demonstrated that profibrogenic actions of Rac1 were mediated by its ability to activate Hh pathway-dependent mechanisms that stimulated myofibroblastic transition of HSCs and enhanced MF-HSC viability. CONCLUSION: These findings demonstrate that interactions between Rac1 and the Hh pathway control the size of MF-HSC populations and have important implications for the pathogenesis of cirrhosis.

Hedgehog-mediated epithelial-to-mesenchymal transition and fibrogenic repair in nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Repair responses define the ultimate outcomes of liver disease. This study evaluated the hypothesis that fibrogenic repair in nonalcoholic fatty liver disease (NAFLD) is mediated by Hedgehog (Hh) pathway activation and consequent induction of epithelial-to-mesenchymal transitions (EMT) in ductular-type progenitors. METHODS: Immature ductular cells were exposed to Sonic hedgehog (Shh) in the presence or absence of the Hh inhibitor cyclopamine to determine whether Hh-pathway activation directly modulates EMT in liver progenitors. Potential biologic correlates of progenitor cell EMT were assessed using mice fed methionine-choline-deficient + ethionine (MCDE) diets with or without cyclopamine. The effects of increased Hh signaling on EMT and fibrogenic repair during diet-induced NAFLD were also compared in wild-type (WT) and Patched haplo-insufficient (Ptc(+/-)) mice. Finally, evidence of Hh-pathway activation and EMT was examined in liver sections from patients with NAFLD. RESULTS: In cultured progenitors, Shh repressed expression of epithelial genes and EMT inhibitors but induced genes that are expressed by myofibroblasts. Cyclopamine reversed these effects. In mouse NAFLD models, Hh-pathway activation, EMT, expansion of myofibroblastic populations, and liver fibrosis occurred. Cyclopamine inhibited Hh-pathway activation and induction of EMT. Ptc(+/-) mice, which have an overactive Hh pathway, exhibited sustained overinduction of Hh target genes and more EMT, myofibroblast accumulation, and fibrosis than WT mice. Numbers of Shh-producing cells and Hh-responsive ductular cells that expressed EMT markers increased in parallel with liver fibrosis in patients with NAFLD. CONCLUSIONS: Hh-mediated EMT in ductular cells contributes to the pathogenesis of cirrhosis in NAFLD.

Identification of a basal-like subtype of breast ductal carcinoma in situ.

Microarray profiling of invasive breast carcinomas has identified subtypes including luminal A, luminal B, HER2-overexpressing, and basal-like. The poor-prognosis, basal-like tumors have been immunohistochemically characterized as estrogen receptor (ER)-negative, HER2/neu-negative, and cytokeratin 5/6-positive and/or epidermal growth factor receptor (EGFR)-positive. The aim of this study was to determine the prevalence of basal-like ductal carcinoma in situ in a population-based series of cases using immunohistochemical surrogates. A total of 245 pure ductal carcinoma in situ cases from a population-based, case-control study were evaluated for histologic characteristics and immunostained for ER, HER2/neu, EGFR, cytokeratin 5/6, p53, and Ki-67. The subtypes were defined as: luminal A (ER+, HER2-), luminal B (ER+, HER2+), HER2 positive (ER-, HER2+), and basal-like (ER-, HER2-, EGFR+, and/or cytokeratin 5/6+). The prevalence of breast cancer subtypes was basal-like (n = 19 [8%]); luminal A, n = 149 (61%); luminal B, n = 23 (9%); and HER2+/ER-, n = 38 (16%). Sixteen tumors (6%) were unclassified (negative for all 4 defining markers). The basal-like subtype was associated with unfavorable prognostic variables including high-grade nuclei (P < .0001), p53 overexpression (P < .0001), and elevated Ki-67 index (P < .0001). These studies demonstrate the presence of a basal-like in situ carcinoma, a potential precursor lesion to invasive basal-like carcinoma.

Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study.

CONTEXT: Gene expression analysis has identified several breast cancer subtypes, including basal-like, human epidermal growth factor receptor-2 positive/estrogen receptor negative (HER2+/ER-), luminal A, and luminal B. OBJECTIVES: To determine population-based distributions and clinical associations for breast cancer subtypes. DESIGN, SETTING, AND PARTICIPANTS: Immunohistochemical surrogates for each subtype were applied to 496 incident cases of invasive breast cancer from the Carolina Breast Cancer Study (ascertained between May 1993 and December 1996), a population-based, case-control study that oversampled premenopausal and African American women. Subtype definitions were as follows: luminal A (ER+ and/or progesterone receptor positive [PR+], HER2-), luminal B (ER+ and/or PR+, HER2+), basal-like (ER-, PR-, HER2-, cytokeratin 5/6 positive, and/or HER1+), HER2+/ER- (ER-, PR-, and HER2+), and unclassified (negative for all 5 markers). MAIN OUTCOME MEASURES: We examined the prevalence of breast cancer subtypes within racial and menopausal subsets and determined their associations with tumor size, axillary nodal status, mitotic index, nuclear pleomorphism, combined grade, p53 mutation status, and breast cancer-specific survival. RESULTS: The basal-like breast cancer subtype was more prevalent among premenopausal African American women (39%) compared with postmenopausal African American women (14%) and non-African American women (16%) of any age (P<.001), whereas the luminal A subtype was less prevalent (36% vs 59% and 54%, respectively). The HER2+/ER- subtype did not vary with race or menopausal status (6%-9%). Compared with luminal A, basal-like tumors had more TP53 mutations (44% vs 15%, P<.001), higher mitotic index (odds ratio [OR], 11.0; 95% confidence interval [CI], 5.6-21.7), more marked nuclear pleomorphism (OR, 9.7; 95% CI, 5.3-18.0), and higher combined grade (OR, 8.3; 95% CI, 4.4-15.6). Breast cancer-specific survival differed by subtype (P<.001), with shortest survival among HER2+/ER- and basal-like subtypes. CONCLUSIONS: Basal-like breast tumors occurred at a higher prevalence among premenopausal African American patients compared with postmenopausal African American and non-African American patients in this population-based study. A higher prevalence of basal-like breast tumors and a lower prevalence of luminal A tumors could contribute to the poor prognosis of young African American women with breast cancer.

AlphaB-crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer.

Recent gene profiling studies have identified a new breast cancer subtype, the basal-like group, which expresses genes characteristic of basal epithelial cells and is associated with poor clinical outcomes. However, the genes responsible for the aggressive behavior observed in this group are largely unknown. Here we report that the small heat shock protein alpha-basic-crystallin (alphaB-crystallin) was commonly expressed in basal-like tumors and predicted poor survival in breast cancer patients independently of other prognostic markers. We also demonstrate that overexpression of alphaB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, alphaB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing alphaB-crystallin by RNA interference inhibited these abnormalities. alphaB-Crystallin overexpression also induced EGF- and anchorage-independent growth, increased cell migration and invasion, and constitutively activated the MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by alphaB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing alphaB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that alphaB-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors.