The Small Molecule Inhibitor QLT-0267 Decreases the Production of Fibrin-Induced Inflammatory Cytokines and Prevents Post-Surgical Peritoneal Adhesions.
ABSTRACT: Peritoneal adhesions develop after abdominal surgery, trauma or intraperitoneal infections, and have important consequences. The deposition of peritoneal fibrin is a common pathophysiological pathway for the formation of adhesions. Here, we aimed to examine the effects of fibrin-induced cytokine production on peritoneal mesothelial cells (PMCs), and to block the effects of fibrin using an integrin-linked kinase (ILK) inhibitor, QLT-0267. PMCs were cultured from the enzymatic disaggregation of rat omentum. After the PMCs were covered with fibrin, the expression of IL-1?, IL-6, TNF? and VEGF-A increased. This increase in cytokine production was attenuated by QLT-0267, which acted via the inhibition of both the ILK and focal adhesion kinase (FAK) pathways, and subsequently via the GSK-3? pathway. We found that QLT-0267 decreased both the severity of peritoneal adhesion and the serum levels of IL-6 in our post-surgical adhesion mouse model. In conclusion, our study provides novel evidence that fibrin-induced cytokine production may involve in the mechanism of peritoneal adhesion formation. Furthermore, the use of the small molecule inhibitor QLT-0267 is a new strategy in preventing peritoneal adhesion in patients undergoing abdominal surgery.
Project description:Integrin-linked kinase (ILK) localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.
Project description:Insulin promotes neuronal survival by activating a phosphatidylinositol 3-kinase (PI 3-kinase)/AKT-dependent signaling pathway and reducing caspase activation. We investigated a role for integrin-linked kinase (ILK) in insulin-mediated cell survival in cultured neurons and differentiated R28 cells. We used a serum and depolarization withdrawal model to induce apoptosis in cerebellar granule neurons and a serum withdrawal model to induce apoptosis in differentiated R28 cells. ILK knock-out decreased insulin-mediated protection as did the addition of pharmacological inhibitors of ILK, KP-392 or QLT-0267. Prosurvival effects of insulin were rescued by Boc-Asp (O-methyl)-CH<sub>2</sub>F (BAF), a pancaspase inhibitor, in the presence of KP-392. Insulin and IGF-1 decreased caspase-3 activation, an effect that was inhibited by KP-392 and QLT-0267. Western blot analysis indicates that insulin-induced stimulation of AKT Ser-473 phosphorylation was decreased after the ILK gene was conditionally knocked-out, following overexpression of AKT-DN or in the presence of QLT-0267. Insulin and IGF-1 stimulated ILK kinase activity in primary neurons and this was inhibited following ILK-DN overexpression. Western blot analysis indicates that insulin exposure upregulated the expression of the cellular inhibitor of apoptosis protein c-IAP2 in an extracellular matrix-dependent manner, an effect blocked by KP-392. These results indicate that ILK is an important effector in insulin-mediated neuroprotection.
Project description:Excessive fibrin deposition in the peritoneum is thought to be involved in the development of encapsulating peritoneal sclerosis (EPS), an important cause of morbidity and mortality in peritoneal dialysis patients. We investigated fibrin-induced epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) as a possible mechanism of fibrin involvement in EPS. In vitro, fibrin overlay of PMCs altered their morphology; increased ?-smooth muscle actin, fibronectin, fibroblast specific protein-1, and ?(v)?(3) integrin expression; and decreased cytokeratin 18 and E-cadherin expression. Fibrin overlay also increased focal adhesion kinase and Src kinase phosphorylation. Fibrin-induced changes were inhibited by treating the cells with ?(v)?(3) integrin antibody or pentoxifylline (PTX). In a rat model, intraperitoneal injection of Staphylococcus aureus and fibrinogen induced severe EPS features, which were attenuated by PTX treatment. PTX-treated rats also showed preserved peritoneal ultrafiltration function and lower concentrations of cytokines than the untreated rats. S. aureus- and fibrinogen-injected rats had higher percentage of cytokeratin-positive cells in the omentum fibrotic tissue than controls; this was also reduced by PTX treatment. Our results suggest that fibrin induces EMT of PMCs by engaging ?(v)?(3) integrin and activating associated kinases. Our EPS animal model showed that fibrin-induced EMT was involved in the pathogenesis of peritoneal fibrosis and was inhibited by PTX.
Project description:Post-operative adhesions are a leading cause of abdominal surgery-associated morbidity. Exposed fibrin clots on the damaged peritoneum, in which the mesothelial barrier is disrupted, readily adhere to surrounding tissues, resulting in adhesion formation. Here we show that resident F4/80<sup>High</sup>CD206<sup>-</sup> peritoneal macrophages promptly accumulate on the lesion and form a 'macrophage barrier' to shield fibrin clots in place of the lost mesothelium in mice. Depletion of this macrophage subset or blockage of CD11b impairs the macrophage barrier and exacerbates adhesions. The macrophage barrier is usually insufficient to fully preclude the adhesion formation; however, it could be augmented by IL-4-based treatment or adoptive transfer of this macrophage subset, resulting in robust prevention of adhesions. By contrast, monocyte-derived recruited peritoneal macrophages are not involved in the macrophage barrier. These results highlight a previously unidentified cell barrier function of a specific macrophage subset, also proposing an innovative approach to prevent post-operative adhesions.
Project description:Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.
Project description:Postoperative adhesion formation often ruins the quality of life or is an obstacle to illnesses with curative operation such as cancer. Previously we demonstrated that interferon-?-promoted fibrin deposition drove postoperative adhesion formation. However, its underlying cellular and molecular mechanisms remain poorly understood. We found that myofibroblasts of the adhesion predominantly expressed signature molecules of mesothelial cells that line the serosa. Microarray analysis revealed IL-6 as a key underlying player, supported by elevated IL-6 levels in the peritoneal fluid of post-laparotomy human subjects. Injured serosa of cecum-cauterized mice also exhibited induction of Il6, which was followed by Tnf, concomitant with rapid accumulation of neutrophils, substantial population of which expressed TGF-?1, a master regulator of fibrosis. Besides, neutrophil-ablated mice showed reduction in induction of the adhesion, suggesting that TGF-?1<sup>+</sup>neutrophils triggered the adhesion. Human neutrophils expressed TGFB1 in response to TNF-? and TNF in response to IL-6. Moreover, anti-IL-6 receptor monoclonal antibody abrogated neutrophil recruitment and adhesion formation. Thus, IL-6 signaling represents a potential target for the prevention of postoperative adhesions.
Project description:Although materials and modern surgical techniques have been developed to suppress postoperative adhesions, adhesion formation can still occur, and thus, a novel effective anti-adhesion drug is greatly needed. In the present study, we explored the efficacy of paeoniflorin treatment against postoperative peritoneal adhesions and examined the anti-oxidative stress and anti-inflammatory properties of PE. Forty-eight male Sprague-Dawley rats were randomly divided into 6 groups for the study: the sham, control, hyaluronan and three concentrations (10, 20 and 40 mg/kg/d) paeoniflorin groups. Abdominal adhesions were created by abrasion of the caecum and its opposite abdominal wall. In the paeoniflorin groups, the rats were administered daily oral doses of paeoniflorin for 7 days. The abdominal cavities of the rats were reopened with a U-shaped incision to macroscopically grade the adhesions. Histologic analysis was performed, and oxidative stress, inflammatory cytokine, collagen fiber degradation and cytokeratin levels were measured. Macroscopic and histopathological measurements revealed that paeoniflorin reduced peritoneal adhesion and inflammation. Notably, treatment with paeoniflorin reduced the protein levels of TGF-?1, IL-6 and COX-2. The collagen fiber fractions were distinctly lower in the PE groups than in the control group. Western blotting analyses showed that paeoniflorin increased MMP-9 and superoxide dismutase-2 protein expression and sharply reduced ?-SMA and COX-2 protein expression. Peritoneal mesothelium cells were more continuous and complete in animals treated with paeoniflorin. Our study suggests that paeoniflorin can be used to ameliorate peritoneal adhesions via anti-oxidative stress and anti-inflammatory actions during the postoperative period.
Project description:Postoperative intra-abdominal adhesion is a very common complication after abdominal surgery. One clinical problem that remains to be solved is to identify an ideal strategy to prevent abdominal adhesions. Keratinocyte growth factor (KGF) has been proven to improve the proliferation of mesothelial cells, which may enhance fibrinolytic activity to suppress postoperative adhesions. This study investigated whether the combined administration of KGF and a sodium hyaluronate (HA) gel can prevent intra-abdominal adhesions by improving the orderly repair of the peritoneal mesothelial cells. The possible prevention mechanism was also explored. The cecum wall and its opposite parietal peritoneum were abraded after laparotomy to induce intra-abdominal adhesion formation. Animals were randomly allocated to receive topical application of HA, KGF, KGF + HA, or normal saline (Control). On postoperative day 7, the adhesion score was assessed with a visual scoring system. Masson's trichrome staining, picrosirius red staining and hydroxyproline assays were used to assess the magnitude of adhesion and tissue fibrosis. Cytokeratin, a marker of the mesothelial cells, was detected by immunohistochemistry. The levels of tissue plasminogen activator (tPA), interleukin-6 (IL-6), and transforming growth factor ?1 (TGF-?1) in the abdominal fluid were determined using enzyme-linked immunosorbent assays (ELISAs). Western blotting was performed to examine the expression of the TGF-?1, fibrinogen and ?-smooth muscle actin (?-SMA) proteins in the rat peritoneal adhesion tissue. The combined administration of KGF and HA significantly reduced intra-abdominal adhesion formation and fibrin deposition and improved the orderly repair of the peritoneal mesothelial cells in the rat model. Furthermore, the combined administration of KGF and HA significantly increased the tPA levels but reduced the levels of IL-6, tumor necrosis factor ? (TNF-?) and TGF-?1 in the abdominal fluid. The expression levels of TGF-?1, fibrinogen and ?-SMA protein and mRNA in the rat peritoneum or adhesion tissues were also down-regulated following the combined administration of KGF and HA. The combined administration of KGF and HA can significantly prevent postoperative intra-abdominal adhesion formation by maintaining the separation of the injured peritoneum and promoting mesothelial cell regeneration. The potential mechanism may be associated with rapid mesothelial cell repair in the injured peritoneum. This study suggests that combined administration of KGF and HA may be a promising pharmacotherapeutic strategy for preventing abdominal adhesions, which is worth further study, and has potential value in clinical applications.
Project description:Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size-exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5β1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.
Project description:Peritoneal metastasis (PM) is a very serious complication of gastrointestinal and gynecological malignancies which is poorly documented. Modified mesothelial cell layer and their microenvironments can favor fibrin deposition for cancer cell adhesion. Scanning and transmission electron microscopy of peritoneal surface and cancer cell clusters from cancer patients was done. Ascites and its impact on mesothelial cells were assessed by cytokine array. Neprilysin, matrix metalloprotease, epithelial mesenchymal transition (EMT) related molecules (E-cadherin, Snail, Slug, Twist, Vimentin and Fibronectin), tissues factor (TF), endothelial protein C receptors (EPCR) were quantified by q-PCR. Fibrin in the simples were stained using anti fibrin F1E1 antibody. Migration ability was assessed by scratch assay. Cell viability and neprilysin activity were analyzed by bioluminescence. Cancer cells-fibrin interaction was investigated by scanning electron microscopy (SEM) and microcinematography (MCG). Mesothelial cells change their morphology after incubation with carcinomatosis peritoneal fluids in vitro. EMT associated with upregulation of neprilysin, matrix metalloproteinase-2, tissue factor and cytokines secretions such as interleukin-6, and 8, hepatocyte growth factor and granulocyte chemotactic protein-2 mRNA and protein were observed. EPCR expression as a natural anticoagulant was decreased. In parallel, carcinomatosis cell clusters extracted from peritoneal fluids were found to be associated with fibrin. Kinetic analysis of cancer cell-fibrin interaction in vitro studied by MCG showed that fiber filaments generated from clots inhibited cancer cell adhesion on fibrin clots. These results indicated that fibrin deposit on the peritoneal surface serve as niches for cancer expansion in carcinomatosis patients.