Several Western guidelines recommend the routine use of pharmacologic thromboprophylaxis for cancer surgery patients to prevent venous thromboembolism (VTE). However, the necessity of routine pharmacologic perioperative thromboprophylaxis in Asian gastric cancer (GC) patients has not been clearly determined. To determine the necessity of routine perioperative pharmacologic thromboprophylaxis in Korean gastric cancer patients, the incidence of postoperative VTE was prospectively evaluated in gastric cancer patients receiving surgery. Among 610 GC patients who had received surgery, 375 patents underwent routine duplex Doppler ultrasonography (DUS) on days 5–12 following surgery to detect VTE and then VTE-related symptoms and signs were checked at 4 weeks after surgery (cohort A). The 235 patients that declined DUS were registered to cohort B and the occurrence of postoperative VTE was retrospectively analyzed. In cohort A, symptomatic or asymptomatic VTE until 4 weeks after surgery was detected in 9 patients . Tumor stage was a significant factor related to VTE development . In multivariate analysis, patients with stage IV had a higher postoperative VTE development  than those with stage I. In cohort B, a low incidence of postoperative VTE was reaffirmed; only one postoperative VTE case (0.4%) was observed. In conclusion, the incidence of postoperative VTE in Korean GC patients was only 2.4%. Risk-stratified applications of perioperative pharmacologic thromboprophylaxis are thought to be more appropriate than the routine pharmacologic thromboprophylaxis in Korean GC patients receiving surgery. Venous thromboembolism (VTE), including extremity deep vein thrombosis (DVT) and pulmonary embolism (PE), is attributed to several risk factors including old age, immobilization, surgery and others Gastric cancer (GC) is particularly prevalent in eastern Asia. According to Western guidelines, all GC patients should receive pharmacologic prophylaxis such as low molecular weight heparin (LMWH) Due to ethnic differences in incidences of VTE between Asian and Western cancer patients, studies focusing on VTE in Asian patients are clearly required. Moreover, as GC is prevalent in Asia, large prospective studies on the incidence of postoperative VTE in Asian GC patients are required to justify risk-stratified application of perioperative pharmacologic thromboprophylaxis.This prospective study, conducted at Seoul National University Bundang Hospital (SNUBH), was carried out to investigate the postoperative incidence of VTE in GC patients. The pharmacologic prophylaxis for VTE in GC patients receiving surgery was not routine clinical practice at SNUBH. Patients who were admitted for GC surgery and met the eligibility criteria were consecutively enrolled between May 2010 and July 2011.Patients with ≥ 20 years-of-age and who had pathologically confirmed adenocarcinoma of the stomach or gastroesophogeal junction were included. All patients received major abdominal cancer surgery for curative or palliative intent. Major surgery was defined as a surgical procedure lasting > 30 minutes In the prospective cohort , all patients underwent duplex and color Doppler ultrasonography (DUS) on lower extremities to screen for DVT regardless of postoperative symptom development. All patient demographics and laboratory data were collected before surgery. A variation of the Elixhauser Comorbidity index was used for comorbidities All cohort A patients underwent a DUS between 5–12 days following GC surgery. The DUS was performed by two experienced radiologists . All imaging was performed using a HDI 5000 ultrasound  equipped with a high-resolution 5–9 MHz linear-array transducer, from the distal 3–4 cm of the external iliac vein to the distal calf veins. DUS included imaging in the transverse and longitudinal planes using both gray-scale and color DUS.DVT was defined when the following conditions were seen: (a) echogenic material within lumen, (b) non-compressibility of the affected vein, or (c) nonvisualized flow in color Doppler imaging A routine postoperative follow-up visit was performed at 4 weeks  following surgery and then every 3–6 months. Symptoms and signs related to VTE were checked during the surgical admission and outpatient clinic follow-up periods. Whenever VTE-related symptoms were clinically suspected, the study protocol guided that a DUS or computed tomography angiography (CT angiography) for low extremities or pulmonary vasculature should be conducted.During the study period, 235 patients who did not want to undergo DUS following surgery were registered to cohort B . These pThe primary objective was to find the incidence of symptomatic or asymptomatic VTE following surgery in cohort A patients. The incidence of postoperative VTE was defined as the cases detected by routine DUS (on days 5–12 after surgery) plus any additional VTE cases detected by DUS or other studies (until 4 weeks after surgery). The secondary objective was to identify risk factors for the development of VTE in this population.We assumed that the actual incidence of postoperative VTE would be approximately 6% and this incidence would be lower than 10%. The calculated sample size was 375 with 80% power and one-sided significance level of 0.025. Although the enrollment of 420 patients was initially planned in consideration for a 10% drop-out rate, this study was completed when 375 cohort A patients were enrolled because there had been no drop-out cases at 4 weeks  after surgery.P-values ≤ 0.05 were considered significant and SPSS software was used . The declaration of Helsinki was followed for this study. The patients in cohort A provided written informed consent prior to this study. The exemption of acquiring written consents from cohort B patients was permitted by the Institutional Review Board (IRB) at SNUBH (IRB study number: B-1002/094-007). This study was approved by IRB and was registered to ClinicalTrials.gov (NCT01345773).Chi-square or linear-by-linear association tests were conducted to compare percentages in cross-tabulations and the t-test was used to compare means. In a multivariate analysis to investigate risk factors for VTE, a logistic regression model was applied. Two-sided Among the 610 patients who met eligibility criteria, 375 were enrolled in cohort A . Patientth day after surgery, asymptomatic PE was incidentally detected in a chest CT performed to evaluate the tumor status before initiating palliative chemotherapy and an asymptomatic proximal DVT was also simultaneously detected by additional DUS. No other VTE case was detected between 4 and 12 weeks after surgery. The further detailed characteristics of VTE events are shown in In cohort A, VTE was detected in 9 patients  within the 4 weeks after surgery. The median time to VTE detection was 7 days . VTE was detected in 8 patients by routine DUS; all 8 DVT events were asymptomatic distal calf vein thrombosis, but one patient had subtle dyspnea and a CT revealed a PE simultaneously. In the remaining one patient, on the 25P = 0.008). Compared with the low VTE incidences in patients with stage I (1.4%) and II/III (2.4%), stage IV patients had 9.7% of VTE incidence. The number of comorbidities showed a borderline significance for developing VTE (P = 0.086). Patients aged ≥ 70 years had higher incidences of postoperative VTE than those aged < 70 years (4.7% vs. 1.5%); however, this was not statistically significant (P = 0.126). Laparoscopic surgery showed a numerically lower VTE incidence than open surgery, but this was not also significant (P = 0.190). Although the surgical outcomes (R0 vs. R1/R2) and extent  were associated with different postoperative VTE incidences (P<0.05), those parameters were clearly correlated with tumor stages. All patients who received palliative surgery without a gastrectomy (N = 6) and the 22 of 26 patients who had postoperative residual tumors (R1/R2 surgery) had stage IV. When 344 patients with stage I to III were separately analyzed, the extent of the surgery did not influence the postoperative VTE incidence .Risk factors for postoperative VTE were analyzed in cohort A patients. The results of univariate analyses are presented in P<0.20 in univariate analyses were included (P = 0.076).In multivariate analysis, the clinical parameters with included . Only thst day after surgery without suspected VTE symptoms or postoperative complications; these 3 patients were referred to nearby hospitals for the further follow-up.Compared with patients in the cohort A, the extent of gastrectomy and surgical procedure (laparoscopic vs. open surgery) showed a different distribution in cohort B patients N = 235, . At 4 weIn the cohort B, symptomatic postoperative VTE did not develop. Only one case of asymptomatic PE in a segmental branch of the right lower lobe pulmonary artery was incidentally found in an abdominal CT performed to evaluate postoperative complications. The PE in this case was spontaneously resolved without treatment .This is the largest prospective study on the incidence of postoperative VTE in GC patients. It demonstrated that postoperative VTE is very rare  in Korean GC patients. As routine pharmacologic prophylaxis is generally considered when the incidence of postoperative VTE is ≥ 10% It has been demonstrated that Asians have a lower incidence of VTE In our study, only the disease stage was predictive of postoperative VTE and the incidence of VTE tended to increase in elderly patients in the multivariate analysis . An advaAlthough GC has been reported to have high risk of VTE development in Western studies Although our study was conducted at a single institution, the situations at other Korean institutions are similar to our institution as most Korean GC patients receive surgery at experienced tertiary high-volume centers In summary, the incidence of postoperative VTE was 2.4% in Korean GC patients and only advanced stage was related to the frequent development of postoperative VTE. Risk-stratified applications of perioperative pharmacologic thromboprophylaxis are thought to be appropriate in Korean GC patients. More prospective studies on the postoperative incidence of VTE in Asian cancer patients are also warranted.


Obstructive sleep apnea (OSA) is a prevalent condition and strongly associated with metabolic disorders. Sleep fragmentation (SF) is a major consequence of OSA, but its contribution to OSA-related morbidities is not known. We hypothesized that SF causes specific perturbations in transcriptional networks of visceral fat cells, leading to systemic metabolic disturbances. We simultaneously profiled visceral adipose tissue mRNA and miRNA expression in mice exposed to 6 hours of SF during sleep, and developed a new computational framework based on gene set enrichment and network analyses to merge these data. This approach leverages known gene product interactions and biologic pathways to interrogate large-scale gene expression profiling data. We found that SF induced the activation of several distinct pathways, including those involved in insulin regulation and diabetes. Our integrative methodology identified putative controllers and regulators of the metabolic response during SF. We functionally validated our findings by demonstrating altered glucose and lipid homeostasis in sleep-fragmented mice. This is the first study to link sleep fragmentation with widespread disruptions in visceral adipose tissue transcriptome, and presents a generalizable approach to integrate mRNA-miRNA information for systematic mapping of regulatory networks. Obstructive sleep apnea (OSA) is a highly prevalent disorder in adults and children The advent of transcriptional profiling using mRNA microarrays has yielded important insights into the function of adipose tissue under various conditions ad libitum access to food and water. At the end of the experimental period, the mice underwent brief anesthesia using carbon dioxide (1–2 min) and were euthanized immediately by cervical dislocation. Abdominal cavity was surgically opened and white  adipose tissue was dissected from the epididymis within <5 min, immediately frozen in liquid nitrogen, and stored at −80°C until RNA extraction. All protocols were approved by the Institutional Animal Care and Use Committee and are in close agreement with the National Institutes of Health Guide in the Care and Use of Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.Male C57BJ/6L mice  of 10 weeks of age were used in this study. These mice were housed in 12-hour light/dark cycle (lights on at 7 a.m. to 7 p.m.) in constant temperature (26±1°C) with Using a rodent model of sleep fragmentation recently developed and validated in our laboratory Total RNA and miRNA were isolated from the same visceral fat adipose tissues of each animal . Total RNA was isolated using automated RNA extraction  and DNase treated according to manufacturer's protocol. miRNA was isolated using miRNeasy Mini kit . The RNA quality and integrity were determined using the Eukaryote Total RNA Nano 6000 LabChip assay (Agilent Technologies) on the Agilent 2100 Bioanalyzer. The quality of miRNA was determined using Agilent Small RNA Kit according to the manufacture's protocol. Both total RNA and miRNA samples were quantified on a Nanodrop 2000 .Whole-genome mouse Agilent microarrays were used to profile visceral fat gene expression during SF and control conditions. Purified total RNA (500 ng) was processed for labeling using the Low RNA Input Fluorescent Linear Amplification Kit . Equal quantities of total RNA were labeled with each reaction containing 50 ng of total RNA and 2 µl (34 pg) of RNA spike-in control. Cyanine 3-labeled CTPs were obtained from PerkinElmer/NEN Life Sciences . Agilent's Low Input Fluorescent Linear Amplification kit was used to generate cRNA for one color as we have previously described Each sample was prepared according to the Agilent's miRNA  using the one-color technique in accordance with the manufacturer's instructions. The samples were profiled on the Agilent Mouse miRNA Microarray (Agilent Technologies), consisting of 60-mer DNA probes synthesized in situ that represent 620 mouse miRNA and 39 viral miRNAs from the Sanger database (Version 10.1), using the one-color technique in accordance with the manufacturer's Instructions. Total RNA including enriched miRNA (100 ng) was dephosphorylated with calf intestine alkaline phosphatase , denatured with dimethyl sulfoxide, and labeled with pCp-Cy3 using T4 RNA ligase . The labeled RNAs were hybridized to Mouse miRNA Microarrays Agilent Technologies . After hybridization and washing, the arrays were scanned with an Agilent microarray scanner using high dynamic range settings as specified by the manufacturer. Microarray results were extracted using Agilent Feature Extraction software (v9.5.3.1) Q-value) threshold of <5% for significance. Since even using a strict FDR cutoff, many pathways and genes were significantly enriched, we limited our analysis to subsets of differentially expressed genes within each gene set that were the primary drivers of the enrichment score, known as the leading edge To determine enriched biologic processes, we applied gene set enrichment analysis (GSEA) to the visceral fat mRNA microarray dataset of mice exposed to SF (n = 8) and controls (n = 8) t-test combined with FDR correction using Q-value (cutoff<5%). Gene targets for differentially expressed miRNAs were computationally predicted using the miRanda algorithm http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/) We identified differentially expressed miRNAs in visceral fat tissue of SF and control mice using a Bayesian implementation of the parametric A genetic network was constructed for the enriched “insulin regulation and diabetes pathways” module based on experimentally verified gene product interactions derived from Ingenuity t) values were averaged and the difference between the 18S Ct and the gene of interest Ct were calculated to calculate the relative expression of the gene of interest using the 2−ΔΔCt method qPCR analysis was performed for selected mRNAs and miRNAs using ABI PRISM 7500 System . The same total RNA was used for both microarray and qPCR experiments. To confirm candidate mRNAs, cDNA was synthesized using one microgram of total RNA using a High-Capacity cDNA Archive Kit . Ribosomal 18S rRNA, was used as a reference gene to normalize the expression ratios. All experiments were performed in triplicates. The cycle number (CmiRNAs selected for confirmation were reverse transcribed with looped microRNA-specific reverse transcriptase (RT) primers  using the TaqMan microRNA mouse assay according to the manufacturer's protocol. Briefly, RT reactions were performed in a volume of 15 µl, and each reaction contained 10 ng of enrich miRNA. RT reactions were performed on a GeneAmpPCR System 9600 . Reactions without addition of reverse transcriptase were performed alongside cDNA synthesis of each sample and used in subsequent procedures to control for potential genomic DNA contamination. All TaqMan assays were run in triplicate on an ABI Prism 7500 using TaqMan Universal PCR Master Mix II without UNG . The qPCR results were normalized against an internal control (RNU6), and then expressed as fold changes (SF relative to control conditions).ad libitum access to water). Blood glucose and insulin levels were measured immediately before glucose injection and the homeostasis model assessment (HOMA) insulin resistance (IR) equation (HOMA-IR index) was calculated Metabolic parameters were measured in a different group of control (n = 8) and SF (n = 7) mice following 6 hours of fasting . The GTT time course experiment was analyzed using two-way ANOVA with time and exposure  as variables. The ANOVA Q-value cutoff <5%  in response to SF in visceral fat cells using a toff <5% . We confQ-value<5%). Biologic modules comprised of up-regulated genes in response to SF included “mitochondrion and oxidative phosphorylation”, “insulin regulation and diabetes pathways”, “lipoprotein biosynthesis and protein lipidation” and “proteasome pathways”. Modules enriched in genes down-regulated during exposure to SF included “fatty acid metabolism and regulation by PPARα”, “glycolytic pathways”, “DNA binding and chromosomal organization”, “cytoskeleton and extracellular matrix”, “Rho and Ras GTPase activity” and “complement pathways”.We applied GSEA to mRNA probesets matching to unique genes and demonstrated that SF induced profound transcriptional perturbations in fat tissue . We confAs outlined in Mlx using qPCR . To assess whether SF is associated with adipose tissue dysfunction, we measured plasma triglyceride levels in mice subjected to 3 weeks of SF and found that chronic sleep disruption resulted in significant elevation of circulating triglycerides . Consistin vivo or in vitro experimental system.In this work, we outlined a simple strategy to integrate data from concurrent mRNA and miRNA profiling of the same tissue. Unlike previous methods based on gene-level statistics, we implemented a gene set enrichment approach because most biologic processes result from coordinated activation of many genes This study has several limitations. Exposure to SF was short (6 hours); future work is required to investigate the chronic effects of SF on adipose tissue and metabolism. Although we confirmed differential expression of several candidates using qPCR, we did not measure differences at the protein level. Furthermore, miRNA targets were identified based on computational predictions and not functionally validated. Finally, our enrichment and network analyses represented the current state of knowledge. Since many new relationships between gene products and pathways will be discovered in the future, the presented network analyses capture a subset of the biologic processes activated in this model.In conclusion, we demonstrate that a short exposure to sleep fragmentation perturbs the transcriptional circuitry of visceral adipocytes and induces metabolic dysregulation in mice. We propose a general strategy to integrate whole-genome mRNA and miRNA profiling, and exploit this method to systematically map genetic networks in fat tissue and identify pathways induced by SF in the context of regulatory miRNAs.


Cancer cells breach the endothelium not only through cell-cell junctions but also via individual endothelial cells (ECs), or transcellular invasion. The underlying EC forms a circular structure around the transcellular invasion pore that is dependent on myosin light chain kinase (MLCK) and myosin II regulatory light chain (RLC) phosphorylation. Here we offer mechanistic insights into transcellular invasive array formation amid persistent tensile force from activated EC myosin. Fluorescence recovery after photobleaching (FRAP) experiments, sarcomeric distance measurements using super-resolution microscopy and electron microscopy provide details about the nature of the myosin II invasion array. To probe the relationship between biomechanical forces and the tension required to maintain the curvature of contractile filaments, we targeted individual actin-myosin fibers at the invasion site for photoablation. We showed that adjacent filaments rapidly replace the ablat11ed structures. We propose that the transcellular circumferential invasion array (TCIA) provides the necessary constraint within the EC to blunt the radial compression from the invading cancer cell. The entry of cancer cells into the bloodstream is a critical point in the metastatic cascade More importantly, metastatic breast cancer cells are capable of breaching the endothelium through two distinct mechanisms: (i) paracellular invasion, wherein cancer cells disrupt the EC border; (ii) transcellular invasion in which the cancer cells penetrate individual ECs without causing cell death in vitro condition), ∼10-fold time lag to the initiation of invasion compared to leukocyte migration, and the demand of high spatio-temporal resolution imaging needed to decipher the underlying mechanisms. As a result, to the best of our knowledge there has only been one other study on the EC cytoskeletal rearrangements that occur during cancer invasion, specifically via the transcellular route Several groups have proposed that cancer cells are capable of mimicking leukocytes when undergoing diapedesis de novo myosin assembly or in situ reorganization? Second, the optical resolution limit had previously prevented our ability to elucidate the architecture of myosin within the TCIA. It is unclear if myosin is arranged in a manner comparable to regular stress fibers. Third, it remains to be elucidated if the invasion event would modulate the myosin turnover rate within the TCIA as compared to stress fibers, a potentially informative indicator for functional and organizational distinction. Fourth, the important structure and function relationship of the TCIA remains unexplored to date. It is interesting to consider how an active contractile system maintains its curvature, as well as how this structure contributes to its function in facilitating transcellular invasion, and vice versa. What would the consequence be if the integrity of the TCIA were locally perturbed?The most damaging intracellular trauma incurred during transcellular invasion is the shear and compressive forces imparted by the transmigrating cells, especially considering the larger size of cancer cells compared to leukocytes. To prevent itself from bursting from within, ECs must devise a counter-mechanism that is viscoelastic yet flexible to withstand transmigration. We have reported that interaction with cancer cells induces localized MLCK activity and contractile function in the underlying EC In this study we address these questions by investigating the (i) the mechanism of TCIA formation, (ii) molecular nature of myosin within the array and (iii) the ability of the TCIA to counteract the compressive forces of the invading cancer cell. Our data reveal that the EC actively reorganizes its stress fibers to circumscribe the invasion pore with concomitant increase in myosin density and stability. Some individual stress fibers also undergo stress-induced fracture and recoil from the invasion site resulting in larger invasion pore. To investigate if the TCIA is maintained by the balance of tumor cell compressive force and EC contractile force, we selectively photoablate individual EC stress fibers immediately flanking the invasion pore and show that the adjacent stress fiber rapidly assumes the position of the displaced filament. Our results implicate the EC cytoskeleton as an important self-protective mechanism and highlight the underlying biomechanics as a key determinant of transendothelial migration.ECs are capable of supporting both paracellular and transcellular invasion  [17]. DuThe mechanism triggering TCIA formation is poorly understood. We hypothesize that the TCIA forms as a reaction to forces generated by the invading cancer cell. If this hypothesis holds true, we should be able to detect an early tumor penetration event in which the EC cytoskeleton does not have sufficient time or appropriate signals to undergo reactionary reorganization. Indeed, we have observed early invasion events where there is lack of myosin-enriched arrays. To gain mechanistic insight into the formation of the TCIA, we performed spinning disk confocal microscopy. Due to toxicity associated with overexpressing and imaging multiple fluorescently tagged proteins in HUVECs, we were unable to simultaneously image the EC borders with fluorescent VE-Cadherin (VE-Cad) for prolonged periods. Transcellular invasion is thus detected by differential interference contrast microscopy through which the EC border can be visualized (not shown). During transcellular diapedesis, transmigratory cells often probe the EC dorsal surface prior to migration The recruitment of EC myosin to circumscribe the invasion pore and the subsequent formation of the TCIA implicate a reactionary remodeling process of EC stress fibers to cope with the intracellular microwound. Our data suggest that the local EC myosin is put under a significant level of stress during transcellular invasion and that there is locally potentiated myosin contraction. This is consistent with our previous results showing increased EC myosin II phosphorylation 1/2, mobile fraction and recovery rates of myosin within stress fibers and the TCIA. 1/2 (p = 0.018) and k value (p = 0.0009). We postulate that the decreased myosin II molecular exchange is the result of reduced myosin off-rate from the stress fibers within the TCIA. Our observation of varied t1/2 of myosin recovery rates is consistent with previous results showing a three fold decrease in myosin turnover rates within peripheral actin when compared to stress fibers To determine if myosin exhibits different kinetics within the TCIA compared to stress fibers in a non-invaded cell, we performed fluorescence recovery after photobleaching (FRAP) experiments to compare myosin turnover rates. The distinct myosin turnover rates motivated us to examine the nature of TCIA myosin arrangement with enhanced resolution. We previously observed contraction of myosin in the vicinity of the invasion pore, but were unable to resolve the high density of myosin structure within the TCIA itself beyond the light diffraction limit using conventional confocal microscopy. Here we turned to super-resolution structured illumination microscopy (SIM) to overcome this hurdle and compared myosin ultrastructure within the TCIA versus the paracellular invasion pore. As shown in In stress fibers, myosin showed significantly more relaxed myosin periodicity with a mean sarcomeric distance of ∼447±14 nm. On the other hand, along paracellular invasion pores, EC myosin exhibited a considerable degree of contraction (mean sarcomeric distance ∼323±5.0 nm). In contrast, the sarcomeric distance within the TCIA, a measurement only now made possible with super-resolution microscopy, registered a significantly contracted state (mean sarcomeric distance ∼296±6.5 nm).The sarcomeric distance within normal stress fibers in non-invaded ECs shows a wide distribution of values and a weak clustering around a median of 350 nm. The large range of stress fiber-related sarcomeric distance was expected because myosin contraction along stress fibers had been revealed to be non-uniform The difference in sarcomeric gaps between the TCIA and the paracellular invasion pores, while statistically significant (p = 0.03), is smaller than expected. However, the mean sarcomeric gap of 296 nm within the TCIA is 25% narrower than the shortest mean distance recorded in the literature To further characterize the architecture of the TCIA, we performed platinum replica transmission electron microscopy The actomyosin network of stress fibers and cortical actin bundles are assembled to physiologically provide tensile strength within EC. Due to the intrinsic isometric tension First, gaps were visible between the invasion array and the cancer cell . Figure Equally striking and reproducible was the absence of circularity in the TCIA. In fact, most arrays showed considerable variance in the local radii of curvature. If the invasion array is a tension-driven structure with stress exerted by the invading cancer cell, then the network cannot tolerate a local perturbation of the balanced forces and will therefore undergo rapid remodeling to compensate for the imbalance in tension. Conversely, if the TCIA was not actively counter-balancing the tension against the invading cancer cell, then a perturbation of a single stress fiber would likely have little to no effect on the TCIA structure. To test this hypothesis, we performed laser micro-photoablation. We specifically severed stress fibers at the site of cancer invasion, resulting in immediate scission of the targeted fibers –S4. The early phase is marked by invasive protrusions of cancer cells or podosomes of leukocytes probing of the endothelial cell active transmigrating phase occurs when the underlying EC begins to envelope the incoming transmigrating cell. In the case of cancer migration, this is prominently marked by a circumferential invasion array comprised of a high density of actin and myosin II surrounding the tumor invasion pore, with active myosin contraction potentiated at least in part by localized MLCK activity et al.late phase of transcellular diapedesis upon the exit of the transmigrating cell, wherein ventral lamellipodia, a newly discovered actin-rich structure, seals the pore or “micro wounds” of the endothelial cell as a form of recuperative response.Transendothelial migration of both cancer cell and leukocytes can be mechanistically divided into three distinct stages as defined by the response of the underlying endothelium. The de novo structure We have previously postulated that the TCIA would be a transiently assembled Detailed sarcomeric measurement using structured illumination microscopy indicates that the EC myosin is highly contracted within the TCIA. In fact, the myosin gap we measured approached the length of a bipolar myosin thick filament In this report, we also probed the relationship of viscoelasticity within a curved structure by laser-targeted photoablation of individual TCIA actomyosin fibers adjacent to the invading cancer cells. We revealed that the curvature of the TCIA was tension driven and that the EC cytoskeletal network would actively maintain the angle of the curvature. There is an apparent transference of tensile loads borne by the pre-ablated fibers to neighboring fibers upon scission. Stress fibers have been shown to possess the characteristics of the Kelvin-Voigt viscoelastic model Strikingly, as shown by the many examples presented in this report and elsewhere et al.Our data extends the notion of robust endothelial self-restorative capacity proposed by Martinelli 2, with the exception of MDA-MB231 cells which were cultured at 37° but with normal atmospheric levels of CO2 and O2. For immunofluorescence experiments ECs were plated on no. 1.5 coverslips  or Matek dishes (P35G-1.5-14-C) coated with purified rat tail collagen.Primary human vascular endothelial cells (HUVEC) were purchased from Lonza (C2519A). Cells were cultured in EGM-2MV media , only passages 2–5 were used. Breast cancer cell MDA-MB231, were cultured in Leibovitz media  supplemented with 10% FBS, and 1% penicillin streptomycin solution. 293A cells were grown in DMEM  supplemented with 10% FBS, and 2% L-glutamine. All cells were cultured at 37° and 5% COpAD-eGFP-RLC, was generated using the pAd-CMV-V5-DEST™ Gateway Vector Kit  according to manufacture's protocol from the original pEGFP-RLC plasmid as previously described Adenovirus was generated according to Invitrogen's Virapower Adenovirus Expression Protocol in 293A cells. To infect endothelial cells 0.5–10 ul of amplified virus was added to 1.5 ml of media in a 35 mm dish in media. After 24 hours the media was replaced and cells were maintained until they became confluent  at which point they were used for invasion assays.2 at 37° for 30 minutes before being transferred to a Tokai Hit heated stage top CO2 incubator on the confocal microscope.Cancer cells were labeled for 20 minutes with CellTrace Violet (1 ul per ml of PBS) , rinsed twice with PBS and then allowed to sit 20 minutes in normal growth media. For timelapse live cell imaging experiments, cancer cells with labeled with PKH26 kit from Sigma according to manufacture's protocol. For imaging the gaps between the cancer cells and ECs, cancer cells were labeled with CellTrace Violet (2 ul per ml of PBS) and ECs were labeled with CSFE (3 ul per ml PBS)  and a non-blocking Dylight 550 conjugated antibody to VE-Cadherin . Cells were trypsinized, washed with media, resuspended in complete EGM-2MV media, and added to the endothelial monolayer. Approximately 1.5–2.5 hours following the addition of the cancer cells, invasions were stopped by fixation (see below). For live cell imaging, cancer cells were trypsinized and added to a GFP-RLC expressing EC monolayer in EGM-2MV media and allowed to attach in a incubator COCells were fixed in 3.7% formaldehyde for 5 minutes, permeabilized with 0.05% NP40 in TBS, and stained with VE-Cadherin and AlexaFluor568 , for 30 minutes at 37°. Samples were mounted in Mowiol with 2.5% Dabco prior to imaging.Laser scanning confocal images were captured using Nikon Elements software on a Nikon A1R confocal microscope using a PlanApo λ100X 1.45 NA objective lens (Nikon). Parameters were set to satisfy the Nyquist criterion. Spinning disk confocal microscopy was performed on an Andor XDI Revolution (Andor Technology). The cells were imaged with a Nikon Plan Apo 100×1.49NA TIRF objective every 4 minutes. Images were acquired using MetaMorph with an Andor Neo cMOS camera.1/2, and k values. Statistical significance between TCIA and stress fibers was determined using an unpaired t-test.Live HUVEC monolayers expressing GFP-RLC and labeled with VE-Cad Dylight 550 were imaged using a Nikon A1R laser scanning confocal microscope with a PlanApo λ100X 1.45 NA objective lens (see above). At least two pre-bleach images of invasions or normal stress fibers were acquired. Photobleaching of GFP- RLC was performed for 5 iterations with 100% 488 nm laser transmission at 1.9 μs pixel dwell. We have conducted numerous prior experiments to confirm that our photobleaching condition will photobleach the targeted zone when observed with maximal pinhole size. Image acquisitions of fluorescence recovery were performed 15–20 seconds following initial FRAP for 5 min to obtain more data points within the linear range of the recovery curve to increase confidence of our k-value calculations. Imaging intervals were then increased to every 45 seconds to assure complete recovery following photobleaching. The intensity within the bleached region and a non-bleached stress fiber (as a control) were first measured using Fiji/ImageJ and then plotted with the FRAPCalc program  to obtain the mobile fraction, t2 incubator on the Nikon A1R microscope. An invasion event was located and an ROI was drawn over single stress fibers in each experiment. The ablation was performed with a 405 nm laser  through a Plan Apo 100×/1.45NA TIRF objective lens (Nikon) on a confocal laser scanning microscope . Full laser power for 2 seconds was used. The sample power  was estimated to be 2.1 mW. This value was derived from (i) actual laser power measurements with no lens present, (ii) actual lens-related transmission reduction measurements provided by Nikon USA, (iii) the size of the lens entrance aperture relative to the laser beam diameter, and (iv) laser beam diameter size derived from curve-fitted data of five Nikon objective lenses. We have avoided using the laser power measurement directly through the objective lens, since by doing so we would be measuring the laser transmission through air. This is because high N.A. lenses (above ∼0.95) will trap a fraction of the laser power due to total internal reflection, thus will under-represent the actual sample power when appropriate fluid immersion is used Invasions were performed as described above on ECs grown on glass bottom Matek dishes. At approximately 90 minutes, cells were transferred to a Tokai Hit heated stage top COA region of interest was drawn lining the pore, the ROI was then analyzed in Fiji/ImageJ using the Shape Descriptor 1u plugin For SIM imaging cells were imaged on a Nikon N-SIM system using a Nikon Plan Apo 100×1.49NA TIRF objective. Images were captured using Nikon Elements software with an Andor iXon3 camera. Z-step size was set to 0.100 um (well within Nyquist criterion). 15 images per slice  were captured and then reconstructed using the Nikon Elements software. To minimize saturated pixels for quantification of the myosin spacing in different structures, we obtained images of the same field with various exposures. Images are displayed as extended depth focused maximum intensity projections. Sarcomeric distances were measured using intensity profiles drawn along the invasion pore. The intensity profiles passed through the center of each myosin head and spacing between peaks on the intensity profile indicated sarcomeric spacing. Statistical significance was determined by performing an ordinary one-way ANOVA in combination with Tukey's multiple comparisons in GraphPad Prism.2O, rinsed 3 times 5 minutes with ddH2O, stained for 20 minutes with 0.2% uranyl acetate in ddH20, rinsed 3 times 5 min in ddH2O. Then samples were dehydrated in 10%, 20%, 40%, 60%, 80% and twice in 100% ethanol, for 5 minutes at each step. They were re-stained with 0.2% uranyl acetate in 100% ethanol for 20 minutes. Afterward, they were rinsed twice for 5 minutes in 100% ethanol and twice for 5 minutes in 100% ethanol that was kept in molecular sieve. Next samples were further dehydrated in a Tousimis Samdri-790 critical point dryer. Dehydrated samples were coated with platinum and then carbon using Bal-Tec Med 020 vacuum evaporator. Coated samples were floated on 30% hydrofluoric acid and then transferred to EM grids. Images were acquired using FEI Tecnai Spirit G2 120kV transmission electron microscope.ECs were plated on no. 1.5 coverslips in 35 mm tissue culture dish and invaded with MDA-MB231 cells as described above. Coverslips were quickly rinsed with PBS to wash out any cells that were still floating in the growth medium. Cells were then extracted for 10 minutes at room temperature with 1% Triton x-100 in PHEM buffer  which contained 5 mM phalloidin (Sigma). Extracted cells were quickly rinsed with PHEM buffer to remove extraction buffer and membrane debris. Samples were then fixed in a mixture of 4% formaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.2 for 20 minutes at room temperature. Following fixation, samples were processed in the following order at room temperature: rinsed 3 times 5 minutes with TBS, stained for 20 minutes in 0.1% tannic acid in ddHFigure S1Paracellular and transcellular invasion of MDA-MB231 cells in HUVEC monolayers. Anti-VE-Cadherin (VE-Cad) immunofluorescence allowed us to distinguish paracellular invasion (with perturbed EC border) and transcellular invasion (with intact EC border). Confocal and orthogonal micrographs of paracellular (A–C) and transcellular invasion (D–F). (A–F) MDA-MB231 (Blue) ECs expressing GFP-RLC (green) stained with VE-Cad (red). Scale bar, 10 μM.(TIF)Click here for additional data file.Movie S1Spinning disk confocal video of paracellular invasion of MDA-MB231 into a HUVEC monolayer corresponding to (AVI)Click here for additional data file.Movie S2Spinning disk confocal video of transcellular invasion of MDA-MB231 into a HUVEC monolayer corresponding to (AVI)Click here for additional data file.Movie S3Serial laser ablations of curved stress fibers during invasion corresponding to (AVI)Click here for additional data file.Movie S4Single laser ablation of linear stress fibers during invasion corresponding to (AVI)Click here for additional data file.Movie S5Laser ablation of a single stress fiber from a resting HUVEC cell expressing GFP-RLC (green) corresponding to (AVI)Click here for additional data file.


Colorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS).In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days.HCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death.Both HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. We demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer. Colorectal cancer (CRC) is a major contributor of cancer-related deaths in the United States in vitro and in vivo assays have been developed to study CRC progression. However, none of these techniques are effective in recapitulating the multi-step dissemination process. We have developed an orthotopic mouse CRC metastasis model that can quantitatively and qualitatively reproduce the metastatic phenotype of the human disease to the liver and/or lungs in an in vivo setting in vitro that HCT cells are growth factor-independent in vivoNumerous in vivo showed a significant difference in their ability to form metastatic deposits. Microarray analysis comparing the primary colon carcinoma arising from the two iso-clonal variants revealed striking differences in their gene signature. In vitro analysis of the two iso-clonal sublines revealed differences in cell survival and motility signaling under GFDS conditions.Comparison of HCT116 and HCT116b cells et al. HCT116 and HCT116b sublines were isolated from a primary tissue culture of a single human colon carcinoma as described by Brattain Packaging cells, 293 GP , were co-transfected with a plasmid encoding VSVG envelope protein and a retroviral vector encoding GFP and the G418 resistance gene using FuGene . The viruses were collected 48 h later and used to infect HCT116 and HCT116b cells. After 48 h, the infected HCT116 and HCT116b cells were selected by treatment with G418 for 5 d. This resulted in a stable transfection.Male athymic BALB/c nude mice between 4–6 weeks of age were acquired from the National Cancer Institute (NCI). Housing for these animals was maintained in a HEPA-filtrated environment within sterilized cages. All animals were subjected to a daily 12 hr light/12-hr dark cycle. All animal procedures were conducted with approval of and in compliance with the Roswell Park Cancer Institute Institutional Animal Care and Use Committee (IACUC).6 cells) were inoculated subcutaneously onto the dorsal surfaces of separate BALB/c nude male mice. Once the xenografts were established, measuring approximately 500 mm3, they were excised and minced into 1 mm3 pieces. Two of these pieces were then orthotopically implanted into the colons of other male BALB/c nude mice as previously reported. Briefly, for operative procedures, animals were anesthetized with isoflurane inhalation. A 1 cm laparotomy was performed, and both the cecum and ascending colon were exteriorized. Using 7× magnification and microsurgical techniques, the serosa was disrupted in two different locations. The 1 mm3 pieces of xenograft were sub-serosally implanted using a 9-0 nylon suture at the two points of serosal disruption. The bowel was then returned to the peritoneal cavity and the abdomen was closed with interrupted 5-0 vicryl sutures.Exponentially growing GFP-labeled HCT116 and HCT116b cells  and xylazine (1 mg/mL) by intraperitoneal injection (0.01 mL/mg) and weekly GFP fluorescence imaging was performed for up to 7 wk. Specifically, GFP fluorescence imaging was performed using a light box illuminated by fiberoptic lighting at 470 nm  using a Retiga EXi color CCD camera . High-resolution images were captured directly using a MS-Windows based PC. Images were visually optimized for contrast and brightness using commercial software . Excitation of GFP in the light box facilitated identification of primary and metastatic disease by direct near-real time visualization of fluorescence in live animals.At approximately 6–8 weeks post-implantation, the animals were euthanized. The colon, including any primary tumor, was harvested in addition to the liver, lungs, and heart. These organs were explanted, imaged, and immediately placed in 10% neutral buffered formalin fixative for 24 h. The tissues were then processed and embedded in paraffin. Slides were then cut for hematoxylin and eosin (H and E).Serial sections were cut to complement the H and E sections and were stained with an IgG1 rabbit polyclonal antibody for Ki-67 . Slides from paraffin embedded tissue blocks were stained according to the Apotag  terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assay kit. The Ki67 and TUNEL assay protocols have been described in details in our previous publications Apoptosis assays were performed to determine the response of HCT116 and HCT116b cells to growth factor deprivation stress (GFDS) Transwell motility assays were performed utilizing 8- µm pore, 6.5 mm polycarbonate transwell filters (Corning Costar Corp.). After trypsinizing the cells, single cell suspensions were seeded in supplemental McCoy's 5A medium in the absence of growth factors onto the upper surface of the filters and allowed to migrate towards McCoy's 5A medium with 10% FBS. After 18 hr incubation, MTT was added to the medium. The cells on the upper surface of the filter were removed with a cotton swab, and the cells that had migrated to the underside of the filter were visualized under the microscope and dissolved in DMSO. Absorbance was read at 570 nm.Cells were lysed in a Tris-HCl based buffer containing 0.5% NP-40 and appropriate protease and phosphate inhibitors as described previously The microarray dataset comparing the HCT116 and HCT116b primary colon tumors has been deposited at the Gene Expression Omnibus (GEO). The accession number to the dataset is GSE44381.Bioinformatics analysis was performed using the ingenuity pathway analysis (IPA) software tool obtained from the UNMC bioinformatics core facility.We compared the clonal variation in metastatic capability between HCT116 cells and its iso-clonal variant HCT116b by utilizing the colonic orthotopic implantation of subcutaneously grown xenografts. The HCT116 animals were euthanized between 6–8 weeks. The HCT 116b were sacrificed on day 63 (9 weeks). The HCT116 animals had to be put down earlier because of tumor burden and animal cachexia in accordance with IACUC regulations. As shown in in vivo. Interestingly, no change in Ki67 staining was observed between HCT116 and HCT116b primary tumors indicating no difference in proliferation rate in vivo between these clonal variants (data not shown).We next characterized the HCT116 and HCT116b primary colonic tumors for their effect on cell survival and proliferation. As indicated in Next we sought to determine the differences in gene expression between HCT116 and HCT116b primary colon carcinoma tumor samples. Transcription profiles of the samples were generated using the Affymetrix HGU133plus2.0 genechips. A heat map dendrogram generated and ranked using the 2-fold up- or down-regulation cut-off is shown in the in vitro in HCT116 and HCT116b human colon carcinoma cells Previously, we examined the effects of nutrient and growth factor deprivation We first performed a DNA fragmentation assay to examine the level of cell death in these cells under nutrient and growth factor deprivation stress (GFDS) 3K) and phospholipase Cγ (PLCγ) pathways which control proliferation and cell survival in colon cancer We next determined the level of phosphorylation under GFDS of major signal transduction pathways such as the mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase Click here for additional data file.Figure S2Gene ontology showing % difference in gene expression in different cellular compartments.(TIF)Click here for additional data file.Table S1List of genes differentially regulated between HCT116 and HCT116b primary colon carcinoma tumor samples.(PDF)Click here for additional data file.


We have developed a mouse model of atherosclerotic plaque regression in which an atherosclerotic aortic arch from a hyperlipidemic donor is transplanted into a normolipidemic recipient, resulting in rapid elimination of cholesterol and monocyte-derived macrophage cells (CD68+) from transplanted vessel walls. To gain a comprehensive view of the differences in gene expression patterns in macrophages associated with regressing compared with progressing atherosclerotic plaque, we compared mRNA expression patterns in CD68+ macrophages extracted from plaque in aortic aches transplanted into normolipidemic or into hyperlipidemic recipients. In CD68+ cells from regressing plaque we observed that genes associated with the contractile apparatus responsible for cellular movement (e.g. actin and myosin) were up-regulated whereas genes related to cell adhesion  were down-regulated. In addition, CD68+ cells from regressing plaque were characterized by enhanced expression of genes associated with an anti-inflammatory M2 macrophage phenotype, including arginase I, CD163 and the C-lectin receptor. Our analysis suggests that in regressing plaque CD68+ cells preferentially express genes that reduce cellular adhesion, enhance cellular motility, and overall act to suppress inflammation. Cardiovascular disease (CVD) is a leading cause of death worldwide Our laboratory has developed a model of plaque regression in which an aortic arch segment containing an atherosclerotic plaque from an apoE−/− mouse In the current study a transcriptome wide analysis of the macrophage-specific changes associated with plaque regression was performed. Microarray assays of mRNA from laser captured CD68+ cells revealed notably different molecular profiles in cells from plaques in atherosclerotic aortic arches transplanted into apoE−/− (progression environment) compared with those from plaques in arches transplanted into WT recipients (regression environment).n = 10) for baseline analyses, and two transplant groups, a group (n = 18) from which aortas would be transplanted into apoE−/− recipient mice, and a group (n = 18) from which aortas would be transplanted into wild type recipient mice. In both cases, recipients were 20 week old male mice on standard chow diet. The surgeries were performed as previously described This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the New York University School of Medicine (Permit Number: A3435-01). ApoE−/− (C57Bl/6) mice were weaned at 1 month and put on a Western Diet (WD) containing 21% fat and 0.15% cholesterol  for 16 weeks. These mice were then divided into three groups: a pre-transplant group . Plasma HDL cholesterol was determined by precipitating non-HDL cholesterol (Wako Diagnostic) and then assaying the remaining HDL cholesterol by means of the Infinity Total Cholesterol Reagent.The pre-transplant and grafted arches were removed, embedded in OCT, and frozen. Serial sections (6 µm thick) were generated using a cryostat. To detect monocyte-derived cells (predominately macrophages and macrophage foam cells), sections were stained for CD68 using rat anti-mouse CD68 . Briefly, sections were incubated for 1 hour at room temperature with the primary antibody, then with biotinylated goat anti-rat Ig for 1 hour, followed by incubation with streptavidin-linked alkaline phosphatase, then developed with substrate, and finally counterstained with haematoxylin. Negative controls were performed with an irrelevant primary antibody. To determine the plaque area, images were digitized, the regions between the endothelial and the medial smooth muscle cell layers were traced, and ImagePro Plus software was used to perform the numerical calculations. A similar approach was taken for the area stained for CD68 or Oil Red O. At least five sections per vessel were analyzed and the mean value used as the summary parameter. The same immunostaining protocol with different primary antibodies was used in order to detect arginase I  or vinculin . To assess the lipid content of the plaques, Oil Red O staining was performed as previously described To isolate CD68+ cells from plaques, LCM was performed with the PixCell II  as previously reported Two rounds of amplification with an ultra low input protocol (ULI) were used to generate cRNA labeled with Cy5 or Cy3 from sample RNA (∼1–5 ng) generated by LCM max-k = 1 C = 1. We used leave-one-out cross-validation (LOOCV) approach to estimate predictive accuracy (area under ROC curve) of molecular signatures. The statistical significance of the produced multivariate signatures was tested with a previously developed protocol for assessing statistical significance of multivariate signatures at 0.05 alpha-level To discover de novo causal pathways of plaque regression and select genes for predictive molecular signatures, we used a multivariate gene selection technique (termed Semi-Interleaved HITON-PC without symmetry correction). HITON-PC was applied with Fisher's Z-test, α = 0.05 and qPCR was used to validate specific genes of interest using 100 pg of total RNA per reaction. The primer and probe sequences are shown in ApoE−/− mice were fed a high fat diet for 16 weeks (“baseline mice”) after which the aortic arches were transplanted into either apoE−/− (progression environment) or WT mice (regression environment) per se were identified as those genes that were differentially expressed in baseline (not transplanted) vs. progression (transplanted) groups, and were removed from the analysis. Then transcripts affected by regression were identified as those differentially expressed in the progression vs. regression group. A fold change of 1.2 was used as a threshold to define 1215 transcripts as the “plaque regression” differential profile  baseline apoE−/− mice , 2) progressing mice (apoE−/− donors to apoE−/− recipients) and 3) regressing mice (apoE−/− donors to WT recipients) using microarray technology . First,  profile .We determined which biological processes were enriched in the plaque regression differential profile by comparing this gene set with gene sets classified by biological function in the public gene set collections Gene Ontology, KEGG, Swissprot and Panther families. This analysis revealed that up-regulated transcripts in the plaque regression differential profile were enriched for contractile proteins  and S4. The most highly up-regulated gene (14 fold) under regression conditions was arginase 1. Up-regulation of this gene was validated by qPCR  and at tTo identify genes that may play a causal role in atherosclerotic plaque regression, we applied a multivariate causal pathway discovery method called HITON-PC, and then used these genes to build multivariate molecular signatures to predict regression and to estimate the accuracy of their predictive capability. This approach distills a large global data set signature  into a smaller local cause and effect predictive signature  We have determined a molecular profile of atherosclerosis regression in a mouse transplant model by performing microarray analysis of plaque CD68+ cells. The profile consisted of 1215 transcripts that were differentially expressed, indicating a large change in the transcriptome of CD68+ cells between the regression and progression environments.The plaque regression differential profile revealed a number of findings: 1) genes associated with the contractile apparatus, which plays a crucial role in cellular movement were among the most highly up-regulated; 2) genes encoding proteins that mediate cellular adhesion, like cadherins and vinculin, were significantly down-regulated; 3) genes encoding histones, cell cycle, cell division and DNA metabolism were also significantly down-regulated, suggesting inhibition of cell division during regression; 4) during regression macrophages display an M2-like state with increased expression of classical markers such as arginase I, CD163 and the C lectin receptor. The last findings led to independent studies that have shown enrichment of M2 macrophage factors in other models of regressing plaques The process that stimulates the elimination of CD68+ cells from regressing plaques has not been fully defined. Our previous studies indicate an important functional role for CCR7 in regulating CD68+ emigration process during regression There is macrophage heterogeneity in atherosclerotic plaques, both M1 (inflammatory) and M2 (anti-inflammatory) macrophages are detectable in lesions Our local causal pathway discovery analysis revealed a molecular signature that could distinguish between the treatment groups with high accuracy. Several genes  were selected by HITON-PC as having a causal role in the regression process. Vinculin-1, downregulated in regression, mediates cell adhesion by attaching actin-based microfilaments to the plasma membrane A study in another mouse model  A limitation of our study is the small amount of total RNA obtained from the laser captured CD68+ cells required a two step amplification process. This procedure amplifies the background noise as well as the biological signal, which is reflected in the high rate of false positives (26%) estimated in the plaque regression differential profile and in some discrepancies observed between qPCR and microarrays  remains open for debate, it is commonly accepted that CD68 is a marker expressed by all of them. Given that the bulk of monocyte-derived cells in the plaque are macrophages and macrophage-foam cells, we selected CD68 as a convenient criterion to use for laser capture. We focused on macrophages, given their central role in the pathogenesis of atherosclerosis, and the ease of isolation by laser capture microdissection.We are also aware that that some of the changes in gene expression appear “counterintuitive” in that the levels of “inflammation-associated” factors are increasing rather than decreasing and thus opposite to the overall trend of less inflammation in the regressing plaque. There are a number of potential reasons for this. For example, it is known that the most efficient resolution of inflammation follows a burst of inflammation In conclusion, taking advantage of a transplant model of atherosclerosis regression we provide evidence that during regression macrophage mRNA expression is dynamic and that these changes may affect the inflammatory, migratory, and metabolic properties of plaque macrophages. The insights gained from this analysis will deepen the understanding of atherosclerosis regression and could ultimately lead to new therapeutic targets against cardiovascular disease.Table S1qPCR assays used to confirm candidate genes.(XLS)Click here for additional data file.Table S2The plaque regression differential profile.(XLS)Click here for additional data file.Table S3Biological processes enriched in up-regulated and down-regulated genes in the plaque regression signature.(XLS)Click here for additional data file.Table S4Genes in selected biological processes.(XLS)Click here for additional data file.


In addition, the effect of adenovirus-mediated decorin expression on human bladder cancer cells in vitro was examined. We first demonstrated using the publicly available GeneSapiens databank that decorin gene expression is present in both normal and malignant human bladder tissues. However, when we applied in situ hybridization with digoxigenin-labeled RNA probes for decorin on human bladder carcinoma tissue samples derived from a large radical cystectomy patient cohort (n = 199), we unambiguously demonstrated that invasive and non-invasive bladder carcinoma cells completely lack decorin mRNA. The cancer cells were also negative for decorin immunoreactivity. Instead, decorin expression was localized solely to original non-malignant stromal areas of bladder tissue. In accordance with the aforementioned results, human bladder cancer cells in vitro were also negative for decorin expression as shown by RT-qPCR analyses. The lack of decorin expression by bladder cancer cells was shown not to be due to the methylation of the proximal promoter region of the decorin gene. When bladder cancer cells were transfected with a decorin adenoviral vector, their proliferation was significantly decreased. In conclusion, we have shown that human bladder cancer cells are totally devoid of decorin expression. We have also shown that adenovirus-mediated decorin gene transduction of human bladder cancer cell lines markedly inhibits their proliferation. Thus, decorin gene delivery offers new potential therapeutic tools in urothelial malignancies.Decorin, a multifunctional small leucine-rich extracellular matrix proteoglycan, has been shown to possess potent antitumour activity. However, there is some uncertainty whether different cancer cells express decorin in addition to non-malignant stromal cells. In this study we clarified decorin expression by human bladder cancer cells both  Today we understand that extracellular matrix (ECM) macromolecules do not only form an inert space filling microenvironment around the cells, but act as a dynamic structure generating signals to control cell behaviour Small leucine-rich proteoglycans (SLRPs) form a gene family of five subclasses consisting of 18 members, including decorin, the prototype member of the family, and its close relative, biglycan The role of decorin in cancer progression and its therapeutic potential as a tumour suppressing antimetastatic agent has been the focus of numerous studies th most common cancer diagnosis worldwide In bladder cancer, which is the 9in vivo and in vitro. We also examined the effect of decorin gene transduction on the proliferation of human bladder cancer cells in vitro.Although several studies have examined decorin expression in various cancers including bladder cancer, there is some uncertainty whether different cancer cells express it or not. In this study, we examined the expression of decorin by human bladder cancer cells both http://www.genesapiens.org/) covers the relative gene expression patterns for 17 330 genes across all the 9783 annotated normal and pathological human tissue samples from publicly available Affymetrix microarray experiments. In GeneSapiens database there is information on 35 different epithelial carcinomas in vivo. For this study, we used decorin expression of 174 samples representing human bladder cancer and compared with data derived from 20 normal human bladder tissue samples.We used GeneSapiens database to evaluate the previously published results regarding decorin expression in normal and malignant human tissues Ethical approval for the use of the clinical material of this study was given by the Ethics Committee of Hospital District of Southwest Finland. The Ethics Committee waived the need for written informed consent. Bladder cancer tissue samples were derived from 199 radical cystectomy patients operated in 1985–2005 in Turku University Hospital, Turku, Finland. The pathological characteristics of the patients are presented in We performed decorin ISH on all TMA sections by probing the samples with human decorin antisense and sense single-stranded RNA riboprobes as previously described in detail The IHC analyses were performed for decorin with a rabbit polyclonal antibody  and for biglycan with a goat polyclonal antibody  on all TMA sections as previously described 2. The cells were trypsinized, pooled, and the RNA was extracted using NucleoSpin RNA II –kit  according to the manufacturer's instructions.Three human urinary bladder cancer cell lines RT-4, 5637, and T24 were purchased from American Type Culture Collection (ATCC). These cell lines were used for RT-qPCR analysis for decorin. All the cell lines were maintained in Dulbecco's modified Eagle medium  containing 10% fetal bovine serum , penicillin (100 IU/ml) and streptomycin (100 µg/ml) , and grown at 37°C with 5% CORNA concentrations from the cancer cell line extractions were determined using a NanoDrop spectrophotometer  and the integrity of the RNA was confirmed with agarose gel electrophoresis. One µg of RNA was DNase treated with RQ1 RNase-Free DNase  and reverse transcribed into cDNA using M-MLV reverse transcriptase and Oligo(dT)15 primer  according to manufacturer's instructions. RT-qPCR was performed as previously described  CAG GTG TGG AAA GGA GGA GG -3′; DCN_A1_R 5′- GTG TCA GCC GGA TTG TGT TC -3′; DCN_A2_F 5′- AGT CCT CAC CTG AAC CCT GA -3′; DCN_A2_R 5′- GAA AGC AGC ATC TTG CCT GG -3′; DCN_B-E _F 5′- CTG CAT CCC ACT CAC CCA AA -3′; DCN_B-E_R 5′- TTC CTG ATG ACC GCG ACT TC -3′. Control loci associated with GAPDH and TSH2B gene promoters (Diagenode) were used as negative and positive controls for DNA methylation, respectively. The recovery % of the methylated DNA was calculated with the formula: recovery % input  = 2? ((Ct input-log input dilution) – CtMeDIP)*100.Total DNA was extracted from urinary bladder cancer cell lines  using QIAamp® genomic DNA kit  following the manufactureŕs protocol. Methylated DNA was enriched with two different assays, first with the automated MethylCap and then with the automated MeDIP assay by using epigenetic sample preparation robot SX-8G IP-Star® (Diagenode). Briefly, the genomic DNA was first fragmented with Covaris S2 sonicator. The methylated DNA fragments were enriched with Methyl Binding Protein Domain (MBD) affinity based MethylCap assay or 5-methylcytosine antibody based MeDIP immunoprecipitation assay as described by the manufacturer (Diagenode). In order to examine the methylation status of decorin gene promoter, the methylated DNA fragments were purified  and subjected to quantitative PCR  by using 7900HT Fast RT-PCR system (Applied Biosystems). Each sample was run in four replicates and three promoter regions covering different decorin isoforms  were examined. The qPCR oligos used in the RT-PCR analysis were: DCN_A1_F 5′-2. The cells were plated on an 8-well chamber slide , 30 000 cells per well. The next day, the cells were transduced with 10, 100 and 1000 pfu/cell of dcn-pxc1c-1 or RAdlacZ in DMEM containing 10 % FBS. 24 hours after transduction, medium was removed and replaced with fresh one. The cells were then grown until the next day, whereafter they were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS). Finally, the proliferation index of decorin transduced cell lines was determined with a Ki-67 rabbit monoclonal antibody  For the transduction experiments, a recombinant replication-deficient adenoviral vector dcn-pxc1c-1 was used as previously described t test was employed in statistical analyses. The p values <0.05 were considered statistically significant.Unpaired Student´s in vivo (data not shown) The GeneSapiens database revealed that decorin is expressed at marked levels in almost all different types of human epithelial carcinoma tissue samples The ISH analyses with DIG-labeled RNA probes for decorin clearly demonstrated that invasive bladder carcinoma cells were totally devoid of decorin mRNA in all bladder cancer tissue samples . The samin vivo results demonstrated that malignant cells within both invasive and non-invasive human bladder cancer tissue samples do not express decorin. Therefore, by using RT-qPCR we next examined whether cell lines representing different grades of human bladder cancer express decorin. The results showed that none of the urinary bladder cancer cell lines, including RT-4 , 5637 (grade II), and T24 (grade III) expressed decorin. In order to elucidate, whether the lack of decorin expression was due to the DNA methylation of the decorin gene promoter, we used two different assays, MeDIP and MethylCap, followed by quantitative RT-PCR to examine the methylation status of the different decorin gene promoter isoforms extracted from the cancer cell lines. Based on these assays we were not able to detect DNA methylation in the decorin gene promoter in any of the bladder cancer cell lines examined (The above examined . The conin vivo or in vitro. Next we examined the effect of targeted decorin transduction on the proliferation of human bladder cancer cells in vitro. We used human bladder cancer cell lines RT4 and T24 and a decorin adenoviral vector for this purpose. The cells were transduced with a titer of 10–1000 pfu/cell of adenoviral vector and a viral concentration of 1000 pfu/cell was chosen for further experiments. The results showed that adenoviral-mediated decorin transduction decreased the proliferation index of the cancer cells with statistical significance (Both the ISH results and the RT-qPCR assays clearly demonstrated that human bladder cancer cells are not able to express decorin either ificance . Also thin vivo and in vitro. First, we evaluated the previous data regarding decorin gene expression in different cancers with special reference to bladder cancer utilizing the publicly available GeneSapiens databank Although decorińs role in inhibiting tumour growth is recognized today in vivo results showed that human bladder cancer cells do not express decorin. This same finding was demonstrated to be also true for human bladder cancer cell lines. Because methylation of the decorin gene has previously been shown to regulate decorin expression in colon cancer Our in vivo e.g. with intravesical therapy, in which cancerous bladder cavity is rinsed with decorin adenovirus containing fluid Studies utilizing decorin transduction have previously been conducted e.g. with breast cancer cells and the results have shown both reduced primary tumour growth and prevention of metastasis in vivo. As we screened the samples for the immunoreactivity for another, very similar SLRP to decorin, namely biglycan, we found that the tumour areas negative for decorin were positive for biglycan immunoreactivity. This indicates that although decorin and biglycan represent highly similar molecules that also share similar functions including their ability to bind TGF-β To the best of our knowledge, the present study is the first to exactly localize decorin mRNA at the cellular level in human bladder cancer in vivo or in vitro, and that decorin expression in malignant human bladder tissue resides merely in the areas of original, non-malignant stroma. We have also shown that the lack of decorin expression by human bladder cancer cells is not due to the methylation of the proximal promoter regions of the decorin gene. Furthermore, we have demonstrated that transduction of cultured human bladder cancer cells with a decorin adenoviral vector causes a significant inhibition in the proliferation of the cells in vitro. Taken together, our results indicate that the lack of decorin expression by bladder cancer cells offers a possibility for using decorin based therapies in human urothelial malignancies.In conclusion, in this study we have shown that human bladder cancer cells do not express decorin either


Interposition of a reversed jejunal loop in short bowel sydrome has previously been investigated in human along with animal models and seemed able to facilitate intestinal adaptation. However, it is unclear if growth hormone and insulin, well known for their implication in short bowel pathophysiology, intervene on this effect.Porcine models were randomly allocated to two cohorts: (1) short bowel (SB) group (n = 8) and (2) short bowel reverse jejunal segment (SB-RS) group (n = 8). Amongst other parameters serum growth hormone and insulin were measured at baseline, as well as on postoperative day 30 and 60.Both endogenous hormones failed to demonstrate significant difference in respect to potential direct effect to mechanisms of enhanced intestinal adaptation in reversed group Regulation of intestinal epithelial loss response is attributed to a complex multistage process with involvement of many different factors  includinA recent meta-analysis has highlighted the promising features of exogenously administered human growth hormone to adult patients with short bowel syndrome . In paraOur most recent article on the subject  consistsAs described previously6, the pigs were randomly allocated to two groups: (1) short bowel (SB) group (n = 8) and (2) short bowel reverse jejunal segment (SB-RS) group (n = 8). Intestinal transit time and nutritional status  evaluation along with histopathological study and immunohistochemical analysis of small bowel tissue specimens were performed. In addition serum Growth Hormone  and Insulin  were measured at baseline, as well as on postoperative day 30 and 60. The differences between the two groups were evaluated with Mann–Whitney-Wilcoxon test for independent samples. Statistical analysis was performed with STATA 8.0 statistical software .No significant differences were demonstrated regarding growth hormone (μΙU/ml) . Similarly, no significant differences were demonstrated regarding insulin (μΙU/ml)  (Table The results suggest a similar outcomes to previous data.The documented difference between two groups, in intestinal growth and proliferation, was attributed at a certain extent on the triggering of the increased decelerated nutrient load -3,6. In . In parallel with the above it is well known that main source of production of both hormones is other than small bowel and secretion is subject to different stimuli and multifarious regulation systems. Indeed, in agreement with the present findings, serum albumin had not exhibited significant changes [The lacks of substantial differences in both factors’ levels appear mainly to reflect the lack of significant weight gain after the interposition in our experimental setting. It may thus be postulated that the histopathologically demonstrated enhanced intestinal adaptation after the interposition  and the  changes .Given these findings on endogenous hormones, the future experimental studies should focus on the effect of exogenously administered growth hormone or insulin upon intestinal adaptation after the interposition of a reversed jejunal segment.SB: Short bowel group; SB-RS: Short bowel reverse jejunal segment group.Michail Papamichail and other co-authors have no conflict of interest.All authors read and approved the final. MP conceived the idea, designed the study and wrote the manuscript. MD conceived the idea, designed the study, revised the manuscript for important intellectual content and has given final approval of the version to be published OP, SL, PP participated in the design of the study, made substantial contributions to the acquisition of data, TNS wrote the manuscript and performed the descriptive statistics.