Close association between nerves and mast cells in the gut wall provides
the microanatomic basis for functional interactions between these elements,
supporting the hypothesis that a mast cell–nerve axis influences gut
functions in health and disease. Advanced morphology and imaging techniques are
now available to assess structural and functional relationships of the mast
cell–nerve axis in human gut tissues. Morphologic techniques including
co-labeling of mast cells and nerves serve to evaluate changes in their
densities and anatomic proximity. Calcium (Ca++) and potentiometric
dye imaging provide novel insights into functions such as mast cell–nerve
signaling in the human gut tissues. Such imaging promises to reveal new ionic or
molecular targets to normalize nerve sensitization induced by mast cell
hyperactivity or mast cell sensitization by neurogenic inflammatory pathways.
These targets include proteinase-activated receptor (PAR) 1 or histamine
receptors. In patients, optical imaging in the gut in vivo has the potential to
identify neural structures and inflammation in vivo. The latter has some risks
and potential of sampling error with a single biopsy. Techniques that image
nerve fibers in the retina without the need for contrast agents (optical
coherence tomography and full-field optical coherence microscopy) may be applied
to study submucous neural plexus. Moreover, the combination of submucosal
dissection, use of a fluorescent marker, and endoscopic confocal microscopy
provides detailed imaging of myenteric neurons and smooth muscle cells in the
muscularis propria. Studies of motility and functional gastrointestinal
disorders would be feasible without the need for full-thickness biopsy.
Mast Cells; Enteric Nervous System; Visceral Afferents; Confocal Microscopy; Tomography
Background & Aims
Paneth cells contribute to the small intestinal niche of Lgr5+ stem cells. Although the colon also contains Lgr5+ stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5+ stem cells.
We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types.
Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117+ crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit+ goblet cells were interdigitated with Lgr5+ stem cells. In vivo, this colonic cKit+ population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit+ cells. When isolated from mouse colon, cKit+ cells promoted formation of organoids from Lgr5+ stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit+ cells using a toxin-conjugated antibody, organoid formation decreased.
cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5+stem cells.
Cancer; Inflammatory Bowel Disease; Intestine; Regenerate
Background & Aims
The type III interferons (IFN-λs: interleukin [IL]-28a, IL-28b, and IL-29) have important roles in hepatitis C virus (HCV) infection, but little is understood about what cells produce these cytokines or how production is activated. We investigated whether human immune cells recognize HCV-infected cells and respond by producing IFN-λ.
We cultured healthy human peripheral blood mononuclear cells (PBMCs) with different populations of immune cells and JFH-1 HCV-infected Huh7.5 (HCVcc/Huh7.5) cells.
Human PBMCs recognized HCVcc/Huh7.5 cells responded by producing IFN-α, IFN-γ, and IFN-λ. A rare subset of myeloid dendritic cells (mDCs), which are BDCA3+, (also called mDC2 cells), were the major source of IL-28 and IL-29 production in response to HCVcc/Huh7.5 cells. Plasmacytoid DCs (pDCs) produced IFN-α, whereas natural killer and natural killer T cells were the main source of IFN-γ production in co-culture experiments. Of the endosomal toll-like receptors (TLRs)3, 7, 8, and 9, only TLR3 or double-stranded HCV RNA induced production of IL-28 and IL-29 by mDC2s; endosomal maturation was required. Production of IFN-α and IFN-λ were linked—IFN-λ increased production of IFN-α by pDCs and IFN-α significantly increased production of IFN-λ.
mDC2s are a major source of IFN-λs production by PBMCs in response to HCVcc/Huh7.5 cells. mDC2s are activated through the TLR3 pathway, indicating that human DCs can efficiently initiate and immune response against HCV infection. IFN-λ therefore has an important role in HCV infection.
IL28B SNP; NK cells, NKT cells; viral immune regulation
Background & Aims
Tumor suppressor proteins are inactivated by many different mechanisms, including nuclear exclusion by chromosome region maintenance (CRM)-1. Increased tumor levels of CRM-1 have been correlated with poor prognosis of patients with pancreatic cancer, making it a therapeutic target. Selective inhibitors of nuclear export (SINEs) bind to CRM-1 to irreversibly inhibit its ability to export proteins; we investigated a new class of SINEs in pancreatic cancer cells.
We studied the effects of SINE analogs in a panel of pancreatic cancer cell lines and non-transformed human pancreatic ductal epithelial (HPDE) cells using proliferation, apoptosis, immunoblot, co-immunoprecipitation, small inhibitor (Si)-RNA, and fluorescence microscopy analyses. The effects of the SINEs were also investigated in mice with subcutaneous and orthotopic tumors.
SINEs (KPT-185, KPT-127, KPT-205 and KPT-227) inhibited proliferation and promoted apoptosis of pancreatic cancer cells, but did not affect HPDE cells. The nuclei of cells incubated with KPT-185 accumulated tumor suppressor proteins (p27, FOXO, p73, and PAR-4) and inhibited interactions between CRM-1 and these proteins. Mutations in the region of CRM-1 that binds to SINEs (Cys-528), or siRNA knockdown of PAR-4, prevented the ability of KPT-185 to block proliferation and induce apoptosis of pancreatic cancer cells. Oral administration of KPT-330 to mice reduced growth of subcutaneous and orthotopic xenograft tumors without major toxicity. Analysis of tumor remnants showed that KPT-330 disrupted the interaction between CRM-1 and PAR-4, activated PAR-4 signaling, and reduced proliferation of tumor cells.
We identified SINEs that inhibit CRM-1 and promote nuclear accumulation of tumor suppressor proteins in pancreatic cancer cells. Oral administration of the drug to mice reduces growth of xenograft tumors.
EXPORTIN; translocation; nucleus; small molecule inhibitor
Background & Aims
We investigated the rate and predictors of anesthesia assistance during outpatient colonoscopy, and whether anesthesia assistance is associated with colonoscopy interventions and outcomes.
We performed a retrospective cohort study using a 20% sample of Medicare administrative claims submitted during the 2003 calendar year. We analyzed data from 328,177 adults, 66 y old or older, over who underwent outpatient colonoscopy examinations.
Overall, 8.7% of outpatient colonoscopies were performed with anesthesia assistance. In multivariate analysis, independent predictors of anesthesia assistance included black race, female sex, and a non-screening indication; anesthesia assistance increased with median income and comorbidities. General and colorectal surgeons, fewer years in their practice, and non-hospital site of service were also significantly associated with anesthesia assistance. The strongest predictor of anesthesia assistance was the Medicare carrier, with odds ratios ranging from 0.22 (95% confidence interval [CI], 0.12–0.43) for the Arkansas carrier (crude rate 0.9%) to 9.90 (95% CI, 7.92–12.39) for the Empire carrier in New York area (crude rate 35.3%), compared with the Wisconsin carrier (crude rate 4.3%). There was also considerable variation amongst endoscopists; 75% of providers had no colonoscopies with anesthesia assistance recorded in their dataset, whereas 4.5% of providers had anesthesia assistance in at least three-quarters of their examinations. Anesthesia assistance was not associated with the diagnosis of polyps, the performance of biopsy or polypectomy, or complications in multivariate analyses.
There are significant variations among regions and sites of service in anesthesia assistance during outpatient colonoscopies of Medicare beneficiaries. Although this variation has considerable economic implications, it was not associated with measures of patient risk or outcomes, such as polyp detection or procedure-related complications.
colorectal cancer screening; cost efficacy; quality; pain
Imaging strategies that detect early-stage esophageal squamous cell carcinoma (ESCC) could improve clinical outcomes, combined with endoscopic approaches. Periostin is an integrin-binding protein that is important in the tumor microenvironment. We created a fluorescent-labeled antibody that recognizes periostin and binds specifically to ESCC xenograft tumors in mice. In L2-cre;p120ctnLoxP/LoxP mice, which develop squamous cell cancers that resemble human ESCC, we visualized the probe in preneoplastic and neoplastic esophageal lesions using near-infrared fluorescent imaging with upper gastrointestinal endoscopy. Periostin might be a biomarker of the esophageal tumor microenvironment that can be used to detect preneoplastic lesions.
mouse model; neoplasm; extracellular matrix; POSTN
BACKGROUND & AIMS
Disordered defecation is attributed to pelvic floor dyssynergia. However, clinical observations indicate a spectrum of anorectal dysfunctions. The extent to which these disorders are distinct or overlap is unclear; anorectal manometry might be used in diagnosis, but healthy persons also can have abnormal rectoanal pressure gradients during simulated evacuation. We aimed to characterize phenotypic variation in constipated patients through high-resolution anorectal manometry.
We evaluated anorectal pressures, measured with high-resolution anorectal manometry, and rectal balloon expulsion time in 62 healthy women and 295 women with chronic constipation. Phenotypes were characterized by principal components analysis of high-resolution anorectal manometry.
Two healthy persons and 71 patients had prolonged (>180 s) rectal balloon expulsion time. A principal components logistic model discriminated healthy people from patients with prolonged balloon expulsion time with 75% sensitivity and a specificity of 75%. Four phenotypes discriminated healthy people from patients with abnormal balloon expulsion times; 2 phenotypes discriminated healthy people from those with constipation but normal balloon expulsion time. Phenotypes were characterized based on high anal pressure at rest and during evacuation (high anal), low rectal pressure alone (low rectal) or low rectal pressure with impaired anal relaxation during evacuation (hybrid), and a short anal high-pressure zone. Symptoms were not useful for predicting which patients had prolonged balloon expulsion times.
Principal components analysis of rectoanal pressures identified 3 phenotypes (high anal, low rectal, and hybrid) that can discriminate among patients with normal and abnormal balloon expulsion time. These phenotypes might be useful to classify patients and increase our understanding of the pathogenesis of defecatory disorders.
Anorectal Test; Dyssynergic Defecation; Anismus; Pelvic Floor Dysfunction
BACKGROUND & AIMS
In patients with hepatitis C virus (HCV) infection, interferon alfa (IFN-α) alters expression of IFN-stimulated genes (ISGs), but little is understood about factors that determine outcomes of therapy. We used a systems biology approach to evaluate the acute response of patients with chronic hepatitis C to IFN-α therapy.
We collected liver biopsy samples from 8 treatment-naïve patients with chronic HCV genotype 1 infection at baseline and 24 hours after treatment with IFN-α-2a (10 MU subcutaneously). Blood samples were collected before and up to 48 hours after administration of IFN-α-2a to measure HCV RNA levels and for gene expression analysis. Patients then received pegylated IFN-α-2a and ribavirin on day 5 of the study; therapy continued for up to 48 weeks.
Based on the kinetics of HCV RNA during the first 12 weeks of therapy, 2 patients were rapid virologic responders, 4 were early virologic responders, and 2 did not respond to therapy (nonresponders). Nonresponders had high pretreatment levels of ISG expression in the liver but not in peripheral blood mononuclear cells. In responders, after administration of IFN-α, intrahepatic ISG expression increased significantly from baseline and was associated with a rapid phase 1 decrease in HCV. We identified distinct hepatic expression and tissue distribution patterns of ISGs that segregated with treatment outcome. Importantly, Kupffer cells were a local source of IFN that promoted basal expression of ISG in hepatocytes of non-responders. This finding was validated in cultured THP1 human macrophages that expressed IFN-β after exposure to viable HCV 2a. When Huh7 K2040 and Huh7 L2198S hepatoma cells were incubated with IFN-α-2a, expression of ISGs peaked by 4 hours and decreased by 72 hours, associated with an increase in level of HCV RNA. This indicates that constitutive exposure to IFN causes hepatoma cells to become tolerant of ISG function.
In patients with chronic HCV infection, IFN production by Kupffer cells might promote innate immune tolerance, characterized by a lack of response to IFN therapy. Strategies to disrupt the virus-host interactions that induce innate immune tolerance should improve therapy.
PBMC; Microarray Analysis; Therapeutic Efficacy; Drug
BACKGROUND & AIMS
Opening of the mitochondrial permeability transition pore (MPTP) causes loss of the mitochondrial membrane potential (ΔΨm) and, ultimately, adenosine triphosphate depletion and necrosis. Cells deficient in cyclophilin D (CypD), a component of the MPTP, are resistant to MPTP opening, loss of ΔΨm, and necrosis. Alcohol abuse is a major risk factor for pancreatitis and is believed to sensitize the pancreas to stressors, by poorly understood mechanisms. We investigated the effects of ethanol on the pancreatic MPTP, the mechanisms of these effects, and their role in pancreatitis.
We measured ΔΨm in mouse pancreatic acinar cells incubated with ethanol alone and in combination with physiologic and pathologic concentrations of cholecystokinin-8 (CCK). To examine the role of MPTP, we used ex vivo and in vivo models of pancreatitis, induced in wild-type and CypD−/− mice by a combination of ethanol and CCK.
Ethanol reduced basal ΔΨm and converted a transient depolarization, induced by physiologic concentrations of CCK, into a sustained decrease in ΔΨm, resulting in reduced cellular adenosine triphosphate and increased necrosis. The effects of ethanol and CCK were mediated by MPTP because they were not observed in CypD−/− acinar cells. Ethanol and CCK activated MPTP through different mechanisms— ethanol by reducing the ratio of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide, as a result of oxidative metabolism, and CCK by increasing cytosolic Ca2+. CypD−/− mice developed a less-severe form of pancreatitis after administration of ethanol and CCK.
Oxidative metabolism of ethanol sensitizes pancreatic mitochondria to activate MPTP, leading to mitochondrial failure; this makes the pancreas susceptible to necrotizing pancreatitis.
Tissue Damage; Ethanol Toxicity; Cell Death; Exocrine Pancreas
The Kras gene is mutated to an oncogenic form in most pancreatic tumors. However, early attempts to use this molecule as a specific biomarker of the disease, or inhibit its activity as a cancer therapy, failed. This left a situation in which everyone was aware of the association between this important oncogene and pancreatic cancer, but no one knew what to do about it. Recent findings have changed this picture—many assumptions made about KRAS and its role in pancreatic cancer were found to be incorrect. Several factors have contributed to increased understanding of the activities of KRAS, including creation of genetically engineered mouse models, which have allowed for detailed analyses of pancreatic carcinogenesis in an intact animal with a competent immune system. Cancer genome sequencing projects have increased our understanding of the heterogeneity of individual tumors. We also have a better understanding of which oncogenes are important for tumor maintenance and are therefore called “drivers.” We review the advances and limitations of our knowledge about the role of Kras in development of pancreatic cancers and the important areas for future research.
Kras; Inflammation; Pancreatic Cancer; Mouse Model
Background & Aims
Photodynamic therapy (PDT) has been shown to be effective in the treatment of high-grade dysplasia (HGD)/mucosal carcinoma in Barrett’s esophagus (BE). Substantial proportions of patients do not respond to PDT or progress to carcinoma despite PDT. The role of biomarkers in predicting response to PDT is unknown. We aimed to determine if biomarkers known to be associated with neoplasia in BE can predict loss of dysplasia in patients treated with ablative therapy for HGD/intramucosal cancer.
Patients with BE and HGD/intramucosal cancer were studied prospectively from 2002 to 2006. Biomarkers were assessed using fluorescence in situ hybridization performed on cytology specimens, for region-specific and centromeric probes. Patients were treated with PDT using cylindric diffusing fibers (wavelength, 630 nm; energy, 200 J/cm fiber). Univariate and multiple variable logistic regression was performed to determine predictors of response to PDT.
A total of 126 consecutive patients (71 who underwent PDT and 55 patients who did not undergo PDT and were under surveillance, to adjust for the natural history of HGD), were included in this study. Fifty (40%) patients were responders (no dysplasia or carcinoma) at 3 months after PDT. On multiple variable analysis, P16 allelic loss (odds ratio [OR], 0.32; 95% confidence interval [CI], 0.10 – 0.96) predicted decreased response to PDT. BE segment length (OR, 0.71; 95% CI, 0.59 – 0.85), and performance of PDT (OR, 7.17; 95% CI, 2.50 –20.53) were other independent predictors of loss of dysplasia.
p16 loss detected by fluorescence in situ hybridization can help predict loss of dysplasia in patients with BE and HGD/mucosal cancer. Biomarkers may help in the selection of appropriate therapy for patients and improve treatment outcomes.
Several single nucleotide polymorphisms (SNPs) have been associated with colorectal cancer (CRC) susceptibility. Most CRCs arise from adenomas, and SNPs might therefore affect predisposition to CRC by increasing adenoma risk. We found that 8 of 18 known CRC-associated SNPs (rs10936599, rs6983267, rs10795668, rs3802842, rs4444235, rs1957636, rs4939827, and rs961253) were over-represented in CRC-free patients with adenomas, compared with controls. Ten other CRC-associated SNPs (rs6691170, rs6687758, rs16892766, rs7136702, rs11169552, rs4779584, rs9929218, rs10411210, rs4813802, and rs4925386) were not significantly associated with adenoma risk. Genetic susceptibility to CRC in the general population is likely to be mediated in part by predisposition to adenomas.
colorectal adenomas; intermediate phenotype; risk factor; colorectal carcinoma
Background & Aims
Nuclear factor (NF)-κB is activated during early stages of pancreatitis. This transcription factor regulates genes that control many cell activities, including inflammation and survival. There is evidence that activation of NF-κB protects against pancreatitis, and in other cases, that it promotes this disease. We compared the effects NF-κB in different mouse models of pancreatitis to understand these complications.
To model constitutive activation of NF-κB, we expressed a transgene that encodes its p65 subunit or the inhibitor of κB kinase (IKK) 2 in pancreatic acinar cells of mice. We analyzed effects on pancreatic tissues and levels of NF-κB target genes in these mice and compared them to mice that did not express transgenic p65 or IKK2 (controls).
Transgenic expression of p65 led to compensatory expression of the inhibitory subunit IKB-α and therefore, no clear phenotype. However, p65 transgenic mice given injections of caerulein, to induce acute pancreatitis, had higher levels of NF-κB activity in acinar cells, greater levels of inflammation, and more severe outcomes than control mice. In contrast, constitutive expression of IKK2 directly increased the activity of NF-κB in acinar cells and induced pancreatitis. Prolonged activity of IKK2 (3 months) resulted in activation of stellate cells, loss of acinar cells, and fibrosis, which are characteristics of chronic pancreatitis. Co-expression of IKK2 and p65 greatly increased the expression of inflammatory mediators and the severity of pancreatitis, compared with control mice.
The level of NF-κB activation correlates with the severity of acute pancreatitis in mice. Longer periods of activation (3 months) lead to chronic pancreatitis. These findings indicate that strategies to inactivate NF-κB might be used to treat patients with acute or chronic pancreatitis.
immune regulation; cytokines; RelA; gene regulation
Transplantation of organs between genetically different individuals of the same species causes a T cell–mediated immune response that, if left unchecked, results in rejection and graft destruction. The potency of the alloimmune response is determined by the antigenic disparity that usually exists between donors and recipients and by intragraft expression of proinflammatory cytokines in the early period after transplantation. Studies in animal models have identified many molecules that, when targeted, inhibit T-cell activation. In addition, some of these studies have shown that certain immunologic interventions induce transplantation tolerance, a state in which the allograft is specifically accepted without the need for chronic immunosuppression. Tolerance is an important aspect of liver transplantation, because livers have a unique microenvironment that promotes tolerance rather than immunity. In contrast to the progress achieved in inducing tolerance in animal models, patients who receive transplanted organs still require nonspecific immunosuppressant drugs. The development of calcineurin inhibitors has reduced the acute rejection rate and improved short-term, but not long-term, graft survival. However, long-term use of immunosuppressive drugs leads to nephrotoxicity and metabolic disorders, as well as manifestations of overimmunosuppression such as opportunistic infections and cancers. The status of pharmacologic immunosuppression in the clinic is therefore not ideal. We review recently developed therapeutic strategies to promote tolerance to transplanted livers and other organs and diagnostic tools that might be used to identify patients most likely to accept or reject allografts.
Regulatory T Cells; Operational Tolerance; Co-stimulation Blockade; Immunosuppression Weaning
Background & Aims
Activated hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver, undergo apoptosis after cessation of liver injury, thereby contributing to the resolution of liver fibrosis. In this study, we investigated whether HSC deactivation constitutes an additional mechanism of liver fibrosis resolution.
HSC activation and deactivation were investigated by single cell PCR and genetic tracking in transgenic mice expressing tamoxifen-inducible CreER under control of the endogenous vimentin promoter (VimCreER).
Single cell quantitative PCR demonstrated activation of virtually the entire HSC population in fibrotic livers, and a gradual decrease of HSC activation during fibrosis resolution, indicating deactivation of HSCs. VimCreER marked activated HSCs, demonstrated by a 6- to 16-fold induction of a membrane-bound green fluorescent protein (mGFP) Cre-reporter following injection of carbontetrachloride (CCl4) in both liver and isolated HSCs, and a shift in localization of mGFP-marked HSCs from perisinusoidal to fibrotic septa. Tracking of mGFP-positive HSCs revealed the persistence of 40–45% of mGFP expression in livers and isolated HSCs 30–45 days after cessation of CCl4, despite normalization of fibrogenesis parameters, thereby confirming reversal of HSC activation. After fibrosis resolution, mGFP expression was observed again in desmin-positive perisinusoidal HSCs; no mGFP expression was detected in hepatocytes or cholangiocytes, thereby excluding mesenchymal-epithelial transition. Notably, reverted HSCs remained in a primed state, with higher responsiveness to profibrogenic stimuli.
In mice, reversal of HSC activation contributes to the termination of fibrogenesis during fibrosis resolution but results in higher responsiveness of reverted HSCs to recurring fibrogenic stimulation.
BACKGROUND & AIMS
Excellent single-center outcomes of neoadjuvant chemoradiation and liver transplantation (LT) for unresectable perihilar cholangiocarcinoma caused the United Network of Organ Sharing (UNOS) to offer a standardized model of end-stage liver disease (MELD) exception for this disease. We analyzed data from multiple centers to determine the effectiveness of this treatment and the appropriateness of the MELD exception.
We collected and analyzed data from 12 large-volume transplant centers in the US who met the inclusion criteria of treating three or more patients with perihilar cholangiocarcinoma using neoadjuvant therapy followed by liver transplantation from 1993–2010 (n=287 total patients). Center-specific protocols and medical charts were reviewed on-site.
The patients completed external radiation (99%), brachytherapy (75%), radio-sensitizing (98%), and/or maintenance chemotherapy (65%). Seventy-one patients dropped out before liver transplantation (rate of 11.5% in 3 months). Intent-to-treat survival was 68% and 53%, 2 and 5 years after therapy, respectively; post-transplantation, recurrence-free survival rates were 78% and 65%, respectively. Patients outside the UNOS criteria (those with tumor mass >3 cm, trans-peritoneal tumor biopsy, or metastatic disease) or with a prior malignancy had significantly shorter survival times (P<.001). There were no differences in outcomes among patients based on differences in operative staging or brachytherapy. Although most patients came from 1 center (n=193), the other 11 centers had similar survival times after therapy.
Patients with perihilar cholangiocarcinoma who were treated with neoadjuvant therapy followed by liver transplantation at 12 US centers had a 65% rate of recurrence-free survival after 5 years, demonstrating this therapy to be highly effective. An 11.5% dropout rate after 3.5 months of therapy indicates the appropriateness of the MELD exception. Rigorous selection is important for the continued success of this treatment.
liver cancer; biliary; hepatic; treatment efficacy
Background & Aims
After liver injury, bone marrow-derived liver sinusoidal endothelial cell progenitor cells (BM SPCs) repopulate the sinusoid as liver sinusoidal endothelial cells (LSECs). After partial hepatectomy, BM SPCs provide hepatocyte growth factor, promote hepatocyte proliferation, and are necessary for normal liver regeneration. We examined how hepatic vascular endothelial growth factor (VEGF) regulates recruitment of BM SPC and their effects on liver injury.
Rats were given injections of dimethylnitrosamine to induce liver injury, which was assessed by histology and transaminase assays. Recruitment of SPCs was analyzed by examining BM SPC proliferation, mobilization to the circulation, engraftment in liver, and development of fenestration (differentiation).
Dimethylnitrosamine caused extensive denudation of LSEC at 24 hours, followed by centrilobular hemorrhagic necrosis at 48 hours. Proliferation of BM SPCs, number of SPCs in the bone marrow, and mobilization of BM SPCs to the circulation increased 2- to 4-fold by 24 hours after injection of dimethylnitrosamine; within 5 days, 40% of all LSEC came from engrafted BM SPC. Allogeneic resident SPCs, infused 24 hours after injection of dimethylnitrosamine, repopulated the sinusoid as LSEC and reduced liver injury. Expression of hepatic VEGF mRNA and protein increased 5-fold by 24 hours after dimethylnitrosamine injection. Knockdown of hepatic VEGF with antisense oligonucleotides completely prevented dimethylnitrosamine-induced proliferation of BM SPCs and their mobilization to the circulation, reduced their engraftment by 46%, completely prevented formation of fenestration after engraftment as LSEC, and exacerbated dimethylnitrosamine injury.
BM SPC recruitment is a repair response to dimethylnitrosamine liver injury in rats. Hepatic VEGF regulates recruitment of BM SPCs to liver and reduces this form of liver injury.
endothelial progenitor cells; toxic hepatitis; animal model; liver damage
Background & Aims
The Notch signaling pathway is activated in leukemia and solid tumors (such as lung cancer), but little is known about its role in liver cancer.
The intracellular domain of Notch was conditionally expressed in hepatoblasts and their progeny (hepatocytes and cholangiocytes) in mice, through Cre expression, under the control of an albumin and α-fetoprotein enhancer and promoter (AFP-NICD). We used comparative functional genomics to integrate transcriptome data from AFP-NICD mice and human hepatocellular carcinoma (HCC) samples (n=683). A Notch gene signature was generated using the nearest template prediction method.
AFP-NICD mice developed HCC with 100% penetrance when they were 12 months old. Activation of Notch signaling correlated with activation of 3 promoters of insulin-like growth factor 2 (Igf2); these processes appeared to contribute to hepatocarcinogenesis. Comparative functional genomic analysis identified a signature of Notch activation in 30% of HCC samples from patients. These samples had altered expression in Notch pathway genes and activation of IGF signaling, despite a low frequency of mutations in regions of NOTCH1 associated with cancer. Blocking Notch signaling in liver cancer cells with the Notch activation signature using γ-secretase inhibitors or by expressing a dominant negative form of MAML reduced their proliferation in vitro.
Notch signaling is activated in human HCC samples and promotes formation of liver tumors in mice. The Notch signature is a biomarker of response to Notch inhibition in vitro.
Genetically engineered mouse model; Notch activation; Gene expression profiling