Rory L. Smoot

miR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma

It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. Although targets and functional roles for a number of microRNAs have been described in cholangiocarcinoma, many additional microRNAs dysregulated in this tumor have not been assigned functional roles. In this study, we identify elevated miR‐25 expression in malignant cholangiocarcinoma cell lines as well as patient samples. In cultured cells, treatment with the Smoothened inhibitor, cyclopamine, reduced miR‐25 expression, suggesting Hedgehog signaling stimulates miR‐25 production. Functionally, miR‐25 was shown to protect cells against TNF‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptosis. Correspondingly, antagonism of miR‐25 in culture sensitized cells to apoptotic death. Computational analysis identified the TRAIL Death Receptor‐4 (DR4) as a potential novel miR‐25 target, and this prediction was confirmed by immunoblot, cell staining, and reporter assays. Conclusion: These data implicate elevated miR‐25 levels in the control of tumor cell apoptosis in cholangiocarcinoma. The identification of the novel miR‐25 target DR4 provides a mechanism by which miR‐25 contributes to evasion of TRAIL‐induced cholangiocarcinoma apoptosis. (HEPATOLOGY 2012)

Sorafenib inhibits signal transducer and activator of transcription‐3 signaling in cholangiocarcinoma cells by activating the phosphatase shatterproof 2

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is one of the key signaling cascades in cholangiocarcinoma (CCA) cells, mediating their resistance to apoptosis. Our aim was to ascertain if sorafenib, a multikinase inhibitor, may also inhibit JAK/STAT signaling and, therefore, be efficacious for CCA. Sorafenib treatment of three human CCA cell lines resulted in Tyr705 phospho‐STAT3 dephosphorylation. Similar results were obtained with the Raf‐kinase inhibitor ZM336372, suggesting sorafenib promotes Tyr705 phospho‐STAT3 dephosphorylation by inhibiting Raf‐kinase activity. Sorafenib treatment enhanced an activating phosphorylation of the phosphatase SHP2. Consistent with this observation, small interfering RNA–mediated knockdown of phosphatase shatterproof 2 (SHP2) inhibited sorafenib‐induced Tyr705 phospho‐STAT3 dephosphorylation. Sorafenib treatment also decreased the expression of Mcl‐1 messenger RNA and protein, a STAT3 transcriptional target, as well as sensitizing CCA cells to tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL)‐mediated apoptosis. In an orthotopic, syngeneic CCA model in rats, sorafenib displayed significant tumor suppression resulting in a survival benefit for treated animals. In this in vivo model, sorafenib also decreased tumor Tyr705 STAT3 phosphorylation and increased tumor cell apoptosis. Conclusion: Sorafenib accelerates STAT3 dephosphorylation by stimulating phosphatase SHP2 activity, sensitizes CCA cells to TRAIL‐mediated apoptosis, and is therapeutic in a syngeneic rat, orthotopic CCA model that mimics human disease. (HEPATOLOGY 2009.)

Durability of portal venous reconstruction following resection during pancreaticoduodenectomy.

Venous resection and reconstruction is becoming more common during pancreaticoduodenectomy (PD). There are multiple options for reconstruction of the mesenteric venous system ranging from primary repair to grafting with autologous or synthetic material. Few studies report on the patency rates and long-term morbidity of these repairs. We sought to describe our experience with venous reconstruction during PD with specific attention to patency and long-term morbidity and mortality. Thrombosis rates of mesenteric venous reconstruction during PD are low, with low associated morbidity. In this retrospective cohort, clinical, operative, and pathologic data were collected from consecutive patients for 1988 through 2003. Graft patency on follow-up imaging studies was determined, and short- as well as long-term morbidity and mortality were recorded. Sixty-four patients underwent PD with venous resection/reconstruction from 1988 through 2003. Mean patient age was 63 years, with pancreatic ductal adenocarcinoma as the pathology in 88%. Reconstruction consisted of primary lateral venorrhaphy in 29 (45%), PTFE graft in 18 (28%), primary end-to-end repair in 13 (20%), and autologous vein graft in 4 (6%). There was one perioperative death (2%). Follow-up imaging to assess patency was available for a mean of 12.2 months postoperatively. Eleven thromboses were diagnosed at a mean of 11.9 months. Three thromboses (5%) were noted within 30 days and full anticoagulation was chosen. Fifty-three percent of patients received anticoagulation with aspirin, warfarin, or clopidogrel based upon surgeon preference. There was no difference in thrombosis rates between those receiving anticoagulation and those who did not (P=0.65). In those patients with thrombosis outside the acute time period, morbidity was limited to ascites in three patients and splenic vein thrombosis with uncomplicated esophageal varices in another patient. Mesenteric venous resection and reconstruction during PD has a high patency rate, and those reconstructions that do thrombose are associated with a low morbidity. The majority of reconstruction thromboses that occurred late were associated with recurrence.

Management of Giant Hemangioma of the Liver: Resection versus Observation

BACKGROUND Management of patients with giant hemangiomas of the liver encounters persistent controversy. Although recent case series suggest a low complication rate with nonoperative management, the classic paradigm of preventive operative resection remains. STUDY DESIGN A retrospective cohort study was conducted of 492 patients with giant hepatic hemangioma (>4 cm in size) diagnosed between 1985 and 2005 at Mayo Clinic Rochester. Long-term outcomes were assessed by patient survey, with a follow-up of 11 ± 6.4 years. RESULTS Of 492 patients, 289 responded to the survey. In the nonoperative group (n = 233), 20% had persistent or new onset of hemangioma-associated symptoms, including potentially life-threatening complications in 2%. In the operative group (n = 56), perioperative complications occurred in 14%, including potentially life-threatening complications in 7%. None of the operative patients had persistent or new onset of hemangioma-associated symptoms after resection of the dominant hemangioma. In group comparison, the rate of adverse events was similar (20% versus 14%; p = 0.45) with an overall low risk for potentially life-threatening complications (2% versus 7%; p = 0.07). Size of hemangiomas was not associated with adverse events in either group. Subjective health status and quality of life at follow-up were similar in both groups (p > 0.54). CONCLUSIONS Clinical observation of patients with giant hemangioma of the liver has a similar rate of complications compared with operative management, but might prevent the need for invasive interventions in some patients. Clinical observation is preferred in most patients and operative treatment should be reserved for patients with severe symptoms or disease-associated complications.

Is early colonoscopy after admission for acute diverticular bleeding needed

OBJECTIVES:Urgent colonoscopy has been proposed for the diagnosis and management of acute colonic diverticular bleeding. Identification of active bleeding and nonbleeding stigmata facilitates diagnosis and endoscopic therapy, but it is unclear whether urgent colonoscopy after presentation increases the diagnostic yield. This study evaluated the association between timing of colonoscopy and diagnostic yield in patients admitted with acute colonic diverticular bleeding.METHODS:Patients admitted for hematochezia and receiving a diagnosis of diverticular hemorrhage were identified using the Mayo Clinic GI Bleeding Team and Emergency Room Admissions Databases for the years 1998–2000. Timing of colonoscopy was determined from the time of admission. Logistic regression analysis was used to assess whether the timing of colonoscopy was associated with an endoscopic finding of active bleeding or nonbleeding stigmata (or both).RESULTS:A diagnosis of definitive or presumptive diverticular bleeding was made in 78 patients (39 men and 39 women, mean age 78 yr, range 49–96 yr). Twelve patients (15%) had active bleeding or stigmata. Colonoscopies were performed a mean of 18 ± 11 h after admission. The association between a definitive diagnosis of acute diverticular bleeding and the timing of colonoscopy was not significant (p > 0.46).CONCLUSION:No significant association is apparent between the timing of colonoscopy after admission and encountering active bleeding or nonbleeding stigmata. Based on these observations, urgent colonoscopy for these patients does not seem advantageous.

A Smac Mimetic Reduces TNF Related Apoptosis Inducing Ligand (TRAIL)-Induced Invasion and Metastasis of Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA) cells paradoxically express tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL), a death ligand that, failing to kill CCA cells, instead promotes their tumorigenicity and especially the metastatic behaviors of cell migration and invasion. Second mitochondria‐derived activator of caspase (smac) mimetics are promising cancer therapeutic agents that enhance proapoptotic death receptor signaling by causing cellular degradation of inhibitor of apoptosis (IAP) proteins. Our aim was to examine the in vitro and in vivo effects of the smac mimetic JP1584 in CCA. Despite JP1584‐mediated loss of cellular inhibitor of apoptosis‐1 (cIAP‐1) and cIAP‐2, TRAIL failed to induce apoptosis in KMCH‐1, TFK‐1, and BDEneu CCA cells; a finding consistent with a downstream block in death signaling. Because cIAP‐1 and cIAP‐2 also promote nuclear factor kappa B (NF‐κB) activation by the canonical pathway, the effect of JP1584 on this signaling pathway was examined. Treatment with JP1584 inhibited TRAIL‐induced NF‐κB activation as well as TRAIL‐mediated up‐regulation of the NF‐κB target gene, matrix metalloproteinase 7 (MMP7). JP1584 also reduced TRAIL‐mediated CCA cell migration and invasion in vitro. Finally, in a syngeneic rat orthotopic CCA model, JP1584 administration reduced MMP7 messenger RNA levels and extrahepatic metastases.

The role of pelvic floor dysfunction and slow colonic transit in adolescents with refractory constipation.

OBJECTIVE:Although pelvic floor dysfunction (PFD) is recognized as a cause of refractory constipation in adults, this diagnosis is not frequently considered in children and adolescents with refractory constipation. The purpose of this study was to examine the symptoms and colonic transit in adolescents with constipation evaluated for a disorder in pelvic floor function.METHODS:Adolescents with refractory constipation who had undergone anorectal manometry (ARM) and balloon expulsion test (BET) were identified by retrospective review of records. Initial symptoms and the clinicians assessment were used to categorize patients by pediatric Rome II criteria, that is, functional constipation (FC), constipation-predominant irritable bowel syndrome (C-IBS) or functional fecal retention (FFR). Results of scintigraphic colonic transit studies were evaluated. A χ2 test was used to assess the association between individual clinical symptoms and Rome II criteria.RESULTS:Sixty-seven adolescents underwent evaluation of pelvic floor function by tests for PFD: BET was abnormal in 42%. There was no underlying disease or alternative diagnosis to account for the constipation in these patients. Among the 41 patients who also underwent scintigraphic colonic transit, 30% had slow transit constipation and 12% had both slow colonic transit and abnormal BET. Patients classified as C-IBS were more likely to report weight loss (p =0.03), bloating (p = 0.04), and incomplete rectal evacuation (p = 0.03).CONCLUSION:Abnormal pelvic floor function and delayed colonic transit are demonstrable as single or combined problems in adolescents with refractory constipation.

Non-canonical Hedgehog signaling contributes to chemotaxis in cholangiocarcinoma

BACKGROUND & AIMS The Hedgehog signaling pathway contributes to cholangiocarcinoma biology. However, canonical Hedgehog signaling requires cilia, and cholangiocarcinoma cells often do not express cilia. To resolve this paradox, we examined non-canonical (G-protein coupled, pertussis toxin sensitive) Hedgehog signaling in cholangiocarcinoma cells. METHODS Human [non-malignant (H69), malignant (HuCC-T1 and Mz-ChA-1)] and rat [non-malignant (BDE1 and NRC), and malignant (BDEneu)] cell lines were employed for this study. A BDE(ΔLoop2) cell line with the dominant-negative receptor Patched-1 was generated with the Sleeping Beauty transposon transfection system. RESULTS Cilia expression was readily identified in non-malignant, but not in malignant cholangiocarcinoma cell lines. Although the canonical Hh signaling pathway was markedly attenuated in cholangiocarcinoma cells, they were chemotactic to purmorphamine, a small-molecule direct Smoothened agonist. Purmorphamine also induced remodeling of the actin cytoskeleton with formation of filopodia and lamellipodia-like protrusions. All these biological features of cell migration were pertussis toxin sensitive, a feature of G-protein coupled (Gis) receptors. To further test the role of Hedgehog signaling in vivo, we employed a syngeneic orthotopic rat model of cholangiocarcinoma. In vivo, genetic inhibition of the Hedgehog signaling pathway employing BDE(ΔLoop2) cells or pharmacological inhibition with a small-molecule antagonist of Smoothened, vismodegib, was tumor and metastasis suppressive. CONCLUSIONS Cholangiocarcinoma cells exhibit non-canonical Hedgehog signaling with chemotaxis despite impaired cilia expression. This non-canonical Hedgehog signaling pathway appears to contribute to cholangiocarcinoma progression, thereby, supporting a role for Hedgehog pathway inhibition in human cholangiocarcinoma.

Understanding endoluminal gastroplications: a histopathologic analysis of intraluminal suture plications.

BACKGROUND Endoluminal gastroplication is used to treat GERD, with modest results. Little is known of the histologic reaction to endosutures. Thus, the histologic response to intraluminal plications at different penetration depths within the gastric wall was studied in an animal model. METHODS Intraluminal gastroplications were performed through a laparotomy in 18 New Zealand rabbits. Three sets of everted plications were placed at different penetration depths in each stomach: submucosa (SMpl), muscularis propria (Mpl), and serosa (Spl). Animals were randomized to survival times of 3, 10, or 60 days (respectively, Groups I, II, and III). Plications were compared with a grade scale for each histologic healing phase and gross inspection. RESULTS Fusion between folds was absent in all groups. Serosa differed from muscularis propria with respect to the proportion of samples with microscopic ischemia (67% vs. 8%; p = 0.015), remaining sutures in Group III (33% vs. 3%; p < 0.05 in a single test of significance, but correction for multiple testing removes this significance), and remaining plications in Group II (96% vs. 54%; p < 0.05 in a single test of significance, but correction for multiple testing removes this significance). All of the total and partial histologic scores for the corresponding healing phase in each group escalated with penetration depth. Overall comparison of the histologic scores showed a significant difference among the plications in the proliferation (Group II, p = 0.004) and maturation (Group II, p = 0.009) phases. Total scores also differed among the plications in Groups II (p < 0.001) and III (p < 0.001). Plications were absent in all of Group III, with Spl resulting only in a flat scar. CONCLUSION Everted intraluminal gastroplications do not result in fusion between folds irrespective of suture-penetration depth. A flat scar is the final outcome and appears proportional to the amount of ischemia, foreign body reaction, and suture depth.

Inferior vena cava filters in trauma patients: efficacy, morbidity, and retrievability.

BACKGROUND Thromboembolic events are potentially devastating sources of morbidity in trauma patients. With increasing experience and the introduction of retrievable devices, there has been a renewed interest in inferior vena cava (IVC) filters in trauma patients. METHODS The records for consecutive trauma patients undergoing IVC filter placement during the years 2001 to 2005 were reviewed, and clinical, demographic, and procedural data were evaluated for associations with thromboembolic events and device complications. RESULTS During the study years, 226 trauma patients had IVC filters inserted, and 140 of these patients (62%) had retrievable IVC filters placed. Six patients (3%) had a pulmonary embolism with the filter in place, and two patients (1%) had a pulmonary embolism after filter removal. The most common complication was thrombosis in 27 patients (12%), with clinically significant thrombus occurring in 15 patients (7%). There was no association between the type of filter (permanent or retrievable) or the brand of retrievable filter and thrombosis. Specific risk factors for thrombosis could not be identified. Retrievable filters were successfully removed in 61% of patients with retrievable filters. Technical success rate was 97% in those patients who underwent attempted removal. Removal was completed at a median of 21 days (range, 2-292 days). CONCLUSIONS Retrievable IVC filters in trauma patients are safe, but complications do occur with thrombosis being the most common. Retrieval has a high technical success rate when attempted. However, a significant number of trauma patients are lost to follow-up and this may impact the utilization of retrievable filters in this patient population.