Adam C. Gower

Statins (HMG-CoA reductase inhibitors) decrease postoperative adhesions by increasing peritoneal fibrinolytic activity.

Objectives:The aims of this study were to determine if statins reduce adhesion formation in vivo and to identify the mechanism of action in vitro. Background:Intraperitoneal adhesions develop in up to 95% of patients following laparotomy. Adhesions are reduced by mechanisms that up-regulate fibrinolysis within the peritoneum. Statins promote fibrinolysis in the cardiovascular system and may play a role in the prevention of adhesions. Methods:Adhesions were induced in rats (n = 102) using our previously described ischemic button model. Rats received vehicle (controls), lovastatin (30 mg/kg), or atorvastatin (30 mg/kg) as a single intraperitoneal dose at the time of laparotomy. Animals were killed and adhesions were quantified at day 7. Peritoneal fluid and tissue were collected at day 1 to measure tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) by real-time PCR and ELISA. To assess the effects of statins on wound healing, burst pressures were measured in anastomoses of the colon. The effects of lovastatin on tPA and PAI-1 production were measured in vitro in human mesothelial cells (HMC) in the presence or absence of mevalonate (MVA), geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), all intermediates in the cholesterol pathway downstream of HMG-CoA. The effect of a Rho protein inhibitor, exoenzyme C3 transferase, on tPA production was also determined. Results:Lovastatin and atorvastatin reduced adhesion formation by 26% and 58%, respectively (P < 0.05), without affecting anastomotic burst pressure. At 24 hours, tPA mRNA levels in peritoneal tissue and tPA activity in peritoneal fluid from lovastatin-treated animals were increased by 57% and 379%, respectively (P < 0.05), while PAI-1 levels were unchanged. HMC incubated with either lovastatin or atorvastatin showed concentration-dependent increases in tPA production and decreases in PAI-1 production (P < 0.05). These lovastatin-induced changes in tPA and PAI-1 production were significantly reversed by the addition of MVA, GGPP, and FPP. The Rho protein inhibitor increased tPA production and rescued tPA production from the inhibitory effect of GGPP. Conclusion:These data suggest that statins administered within the peritoneum can up-regulate local fibrinolysis, while the in vitro studies show that this effect may be mediated, in part, by intermediates of the cholesterol biosynthetic pathway that regulate Rho protein signaling.

Characterizing the Impact of Smoking and Lung Cancer on the Airway Transcriptome Using RNA-Seq

Cigarette smoke creates a molecular field of injury in epithelial cells that line the respiratory tract. We hypothesized that transcriptome sequencing (RNA-Seq) will enhance our understanding of the field of molecular injury in response to tobacco smoke exposure and lung cancer pathogenesis by identifying gene expression differences not interrogated or accurately measured by microarrays. We sequenced the high-molecular-weight fraction of total RNA (>200 nt) from pooled bronchial airway epithelial cell brushings (n = 3 patients per pool) obtained during bronchoscopy from healthy never smoker (NS) and current smoker (S) volunteers and smokers with (C) and without (NC) lung cancer undergoing lung nodule resection surgery. RNA-Seq libraries were prepared using 2 distinct approaches, one capable of capturing non-polyadenylated RNA (the prototype NuGEN Ovation RNA-Seq protocol) and the other designed to measure only polyadenylated RNA (the standard Illumina mRNA-Seq protocol) followed by sequencing generating approximately 29 million 36 nt reads per pool and approximately 22 million 75 nt paired-end reads per pool, respectively. The NuGEN protocol captured additional transcripts not detected by the Illumina protocol at the expense of reduced coverage of polyadenylated transcripts, while longer read lengths and a paired-end sequencing strategy significantly improved the number of reads that could be aligned to the genome. The aligned reads derived from the two complementary protocols were used to define the compendium of genes expressed in the airway epithelium (n = 20,573 genes). Pathways related to the metabolism of xenobiotics by cytochrome P450, retinol metabolism, and oxidoreductase activity were enriched among genes differentially expressed in smokers, whereas chemokine signaling pathways, cytokine–cytokine receptor interactions, and cell adhesion molecules were enriched among genes differentially expressed in smokers with lung cancer. There was a significant correlation between the RNA-Seq gene expression data and Affymetrix microarray data generated from the same samples (P < 0.001); however, the RNA-Seq data detected additional smoking- and cancer-related transcripts whose expression was were either not interrogated by or was not found to be significantly altered when using microarrays, including smoking-related changes in the inflammatory genes S100A8 and S100A9 and cancer-related changes in MUC5AC and secretoglobin (SCGB3A1). Quantitative real-time PCR confirmed differential expression of select genes and non-coding RNAs within individual samples. These results demonstrate that transcriptome sequencing has the potential to provide new insights into the biology of the airway field of injury associated with smoking and lung cancer. The measurement of both coding and non-coding transcripts by RNA-Seq has the potential to help elucidate mechanisms of response to tobacco smoke and to identify additional biomarkers of lung cancer risk and novel targets for chemoprevention. Cancer Prev Res; 4(6); 803–17. ©2011 AACR.

Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer.

Smoking is the most important known risk factor for the development of lung cancer. Tobacco exposure results in chronic inflammation, tissue injury, and repair. A recent hypothesis argues for a stem/progenitor cell involved in airway epithelial repair that may be a tumor-initiating cell in lung cancer and which may be associated with recurrence and metastasis. We used immunostaining, quantitative real-time PCR, Western blots, and lung cancer tissue microarrays to identify subpopulations of airway epithelial stem/progenitor cells under steady-state conditions, normal repair, aberrant repair with premalignant lesions and lung cancer, and their correlation with injury and prognosis. We identified a population of keratin 14 (K14)-expressing progenitor epithelial cells that was involved in repair after injury. Dysregulated repair resulted in the persistence of K14+ cells in the airway epithelium in potentially premalignant lesions. The presence of K14+ progenitor airway epithelial cells in NSCLC predicted a poor prognosis, and this predictive value was strongest in smokers, in which it also correlated with metastasis. This suggests that reparative K14+ progenitor cells may be tumor-initiating cells in this subgroup of smokers with NSCLC.

Practical Limitations of Bioresorbable Membranes in the Prevention of Intra-Abdominal Adhesions

IntroductionIntra-abdominal adhesions are a significant source of postoperative morbidity. Bioresorbable barriers composed of hyaluronic acid and carboxymethylcellulose (HA/CMC) reduce adhesion formation by physically separating injured or healing peritoneal surfaces. To assess whether the efficacy of a physical barrier can extend beyond the site of application, we evaluated the effectiveness of an HA/CMC barrier in preventing adhesions distal to the site of placement.MethodsAdhesions were induced in rats by creating peritoneal ischemic buttons on either side of a midline incision. An HA/CMC barrier (Seprafilm™ Genzyme) was intraoperatively placed either under the midline incision, unilaterally over half the ischemic buttons, or bilaterally over all ischemic buttons. Control buttons received no HA/CMC. On dayxa07 adhesions were scored. In similar experiments, peritoneal fluid was collected at 24xa0h to assess the effects of HA/CMC on tissue plasminogen activator activity.ResultsPlacement of HA/CMC under the midline incision did not reduce adhesion formation to distal ischemic buttons (72u2009±u20097%) compared to controls (80u2009±u20098%). Unilateral placement of HA/CMC significantly (pu2009<u20090.05) reduced adhesion formation to those ischemic buttons over which the barrier was applied (35u2009±u20097%) compared to both contralateral (83u2009±u20099%) and control (80u2009±u20098%) ischemic buttons. The bilateral application of HA/CMC also significantly (pu2009<u20090.05) reduced adhesion formation to all ischemic buttons compared to controls (22u2009±u20097% vs. 66u2009±u20097%, respectively). HA/CMC did not affect peritoneal tPA activity.ConclusionsEffective adhesion reduction by the physical barrier HA/CMC appears to be limited to the site of application in this rat model. Despite the presence of a bioresorbable membrane at predicted sites of adhesion formation in the peritoneal cavity, adhesions readily form to distal unprotected sites.

Transcriptomic Studies of the Airway Field of Injury Associated with Smoking-Related Lung Disease

The field of injury hypothesis proposes that exposure to an inhaled insult such as cigarette smoke elicits a common molecular response throughout the respiratory tract. This response can therefore be quantified in any airway tissue, including readily accessible epithelial cells in the bronchus, nose, and mouth. High-throughput technologies, such as whole-genome gene expression microarrays, can be employed to catalog the physiological consequences of such exposures in the airway epithelium. Pulmonary diseases such as chronic obstructive pulmonary disease, lung cancer, and asthma are also thought to be associated with a field of injury, and in patients with these diseases, airway epithelial cells can be a useful surrogate for diseased tissue that is often difficult to obtain. Global measurement of mRNA and microRNA expression in these cells can provide useful information about the molecular pathogenesis of such diseases and may be useful for diagnosis and for predicting prognosis and response to therapy. In this review, our aim is to summarize the history and state of the art of such transcriptomic studies in the human airway epithelium, especially in smoking and smoking-related lung diseases, and to highlight future directions for this field.

An FDA Approved Neurokinin-1 Receptor Antagonist is Effective in Reducing Intraabdominal Adhesions when Administered Intraperitoneally, But Not Orally

R Lim, J Morrill, S G Prushik, K L Reed, A C Gower, S E Leeman, A F Stucchi, J M Becker (2008) An FDA Approved Neurokinin-1 Receptor Antagonist is Effective in Reducing Intraabdominal Adhesions when Administered Intraperitoneally, But Not Orally. Journal of Gastrointestinal Surgery 12:1754–1761. DOI:10.1007/s11605-008-0634-4. This article was incorrectly published as a 2008 SSAT Plenary Presentation. The Discussion published with it does not belong with that article, but instead belongs to the article by Lim, et al (DOI:10.1007/s11605-008-0724-3) appearing in this issue. J Gastrointest Surg (2009) 13:43 DOI 10.1007/s11605-008-0767-5

Replication of association between ELAVL4 and Parkinson disease: the GenePD study.

Genetic variants in embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4) have been reported to be associated with onset age of Parkinson disease (PD) or risk for PD affection in Caucasian populations. In the current study we genotyped three single nucleotide polymorphisms in ELAVL4 in a Caucasian study sample consisting of 712 PD patients and 312 unrelated controls from the GenePD study. The minor allele of rs967582 was associated with increased risk of PD (odds ratioxa0=xa01.46, nominal P valuexa0=xa00.011) in the GenePD population. The minor allele of rs967582 was also the risk allele for PD affection or earlier onset age in the previously studied populations. This replication of association with rs967582 in a third cohort further implicates ELAVL4 as a PD susceptibility gene.

A neurokinin-1 receptor antagonist that reduces intraabdominal adhesion formation increases peritoneal matrix metalloproteinase activity

Adhesions remain a significant complication of abdominal surgery. There is a growing body of evidence suggesting that remodeling of peritoneal extracellular matrix by matrix metalloproteinases (MMPs) is involved in adhesion formation. We have shown that administration of a specific neurokinin‐1 receptor (NK‐1R) antagonist (CJ‐12,255, Pfizer) to rats within 5 hours of surgery reduces intraabdominal adhesion formation. Because substance P (SP), the primary NK‐1R ligand, is known to augment tissue fibrosis, the aim of this study was to determine the effects of NK‐1R antagonist administration on peritoneal MMP expression and activity 24 hours after surgery in a rat adhesion model. Following laparotomy, four ischemic buttons were created on the peritoneum of rats that received either an intraperitoneal NK‐1R antagonist or a vehicle at surgery. Adhesion formation was assessed 7 days later. Peritoneal fluid and tissue were collected at 24 hours to assess total MMP activity, as well as MMP‐2, MMP‐8, and MMP‐9 activity. Specific MMP and tissue inhibitors of MMP mRNAs were measured, and the effects of SP on MMP‐3 expression were determined in Met‐5A cells, a human peritoneal mesothelial cell line. NK‐1R antagonist administration reduced adhesion formation by 47% (p<0.05) at 7 days and significantly increased the total MMP activity in peritoneal fluid at 24 hours. There was an accompanying increase (p<0.05) in MMP‐8 and MMP‐9 mRNA expression and activity in peritoneal tissue and fluid, respectively. MMP‐3 mRNA was also increased in the 24‐hour peritoneal tissue, and exposure of Met‐5A cells to SP reduced MMP‐3 expression and activity. These data support a role for MMPs, specifically MMP‐3, MMP‐8, and MMP‐9, in intraabdominal adhesion formation and suggest that the NK‐1R antagonist may reduce adhesions, in part, by increasing MMP activity in the peritoneum by 24 hours after surgery.

Open adhesiolysis is more effective in reducing adhesion reformation than laparoscopic adhesiolysis in an experimental model

This study compared adhesion reformation after open and laparoscopic adhesiolysis in a rat model.

Stasis Predisposes Ileal Pouch Inflammation in a Rat Model of Ileal Pouch-Anal Anastomosis

BACKGROUNDnWhile restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) has become the definitive surgical treatment for patients suffering from chronic ulcerative colitis (CUC), pouchitis still remains a major late complication. Fecal stasis has been implicated in the etiology of ileal inflammation; however, the mechanism(s) remain unclear, in part due to the lack of an animal model. Our goal was to surgically mimic the IPAA procedure in a rat to investigate the hypothesis that stasis leads to biochemical changes that predispose the ileal pouch to inflammation.nnnMATERIALS AND METHODSnThirty-two Sprague-Dawley rats underwent total colectomy with either straight ileorectal (IRA) or IPAA, and 11 nonoperated rats served as controls (Controls). Twenty-one d postoperatively, 48 h serial barium radiographs and 12 h charcoal transit follow-through studies were performed. Following sacrifice, ileal tissue was harvested for the measurement of myeloperoxidase activity (MPO) activity, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) mRNA levels, and histology.nnnRESULTSnSerial barium radiographs showed stasis in the ileal pouch compared with IRA animals, and charcoal transit times that were two times longer (P ≤ 0.05) than that in the straight IRA rats. Ileal pouch MPO levels were significantly elevated in the IPAA rats compared with the straight IRA rats. ICAM-1 and VCAM-1 mRNA levels were not associated with neutrophil infiltration.nnnCONCLUSIONSnThese studies showed that ileal pouch stasis predisposes biochemical and histological evidence of ileal pouch mucosal inflammation. Studies such as this may provide the rationale for novel, adjunct therapies for the management of pouchitis in patients having undergone IPAA for CUC.