Endothelin-1 drives invadopodia and interaction with mesothelial cells through ILK.

Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ETAR) drives invadopodia by a not fully explored coordinated function of ß-arrestin1 (ß-arr1). Here, we report that ß-arr1 links the integrin-linked kinase (ILK)/ßPIX complex to activate Rac3 GTPase, acting as a central node in the adhesion-based extracellular matrix (ECM) sensing and degradation. Downstream, Rac3 phosphorylates PAK1 and cofilin and promotes invadopodium-dependent ECM proteolysis and invasion. Furthermore, ETAR/ILK/Rac3 signaling supports the communication between cancer and mesothelial cells, favoring SOC cell adhesion and transmigration. In vivo, ambrisentan, an ETAR antagonist, inhibits the adhesion and spreading of tumor cells to intraperitoneal organs, and invadopodium marker expression. As prognostic factors, high EDNRA/ILK expression correlates with poor SOC clinical outcome. These findings provide a framework for the ET-1R/ß-arr1 pathway as an integrator of ILK/Rac3-dependent adhesive and proteolytic signaling to invadopodia, favoring cancer/stroma interactions and metastatic behavior.

Dipeptidyl peptidase 4 promotes peritoneal fibrosis and its inhibitions prevent failure of peritoneal dialysis.

Peritoneal dialysis (PD) possesses multiple advantages for end stage renal disease. However, long-term PD triggers peritoneal fibrosis (PF). From the nationwide analysis of diabetic PD patients (n = 19,828), we identified the incidence of PD failure was significantly lower in diabetic patients treated with dipeptidyl peptidase 4 (DPP4) inhibitors. Experimental study further showed high concentration of glucose remarkably enhanced DPP4 to promote epithelial-mesenchymal transition (EMT) in the mesothelial cells. In chlorhexidine gluconate (CG)-induced PF model of rats, DPP4 expression was enriched at thickening peritoneum. Moreover, as to CG-induced PF model, DPP4 deficiency (F344/DuCrlCrlj strain), sitagliptin and exendin-4 treatments significantly inhibited DPP4 to reverse the EMT process, angiogenesis, oxidative stress, and inflammation, resulting in the protection from PF, preservation of peritoneum and the corresponding functional integrity. Furthermore, DPP4 activity was significantly correlated with peritoneal dysfunction. Taken together, DPP4 caused peritoneal dysfunction/PF, whereas inhibition of DPP4 protected the PD patients against PD failure.

Loss of BAP1 Expression in Atypical Mesothelial Proliferations Helps to Predict Malignant Mesothelioma.

Distinguishing reactive mesothelial proliferation from malignant mesothelioma (MM) can be difficult, particularly on small biopsies. In this scenario, a diagnosis of atypical mesothelial proliferation might be rendered. However, the distinction between a reactive process and MM is important for prognosis and treatment. Recently, loss of BRCA1-associated protein 1 (BAP1) expression and/or homozygous deletion of CDKN2A were identified in some MM, but not in reactive mesothelial proliferations. We studied 34 cases of atypical mesothelial proliferation from our institutional files (1993 to 2016) for BAP1 expression, deletion of CDKN2A, and clinical outcome. Fifteen of 34 patients (44%) were subsequently diagnosed with MM. BAP1 expression was lost in 6 of these 15 (40%) patients. Ten of 15 (67%) patients died of disease within a median time of 18.2 months. BAP1 expression was also lost in 1 case of probable MM. In this case atypical mesothelial proliferation was identified in the pleura during a lobectomy procedure for lung adenocarcinoma. Follow-up of 57.0 months was remarkable for visceral and parietal pleural thickening with continued unilateral effusion identified on imaging studies but no subsequent definitive diagnosis of MM. CDKN2A studies by fluorescence in situ hybridization (performed in 31 cases) found no homozygous deletion of that gene in any case. In conclusion, loss of BAP1 expression in atypical mesothelial proliferation helps to predict MM and is a useful adjunct test in these cases. Homozygous deletion of CDKN2A in mesothelial cell proliferations did not prove to be useful to predict MM in cases of atypical mesothelial proliferation.

IL-6 promotes epithelial-to-mesenchymal transition of human peritoneal mesothelial cells possibly through the JAK2/STAT3 signaling pathway.

Long-term peritoneal dialysis (PD) therapy results in functional and structural alteration of the peritoneal membrane, including epithelial-to-mesenchymal transition (EMT). Interleukin 6 (IL-6) is a local pleiotropic cytokine, hypothesized to play an important role in EMT. This study was designed to investigate the role of IL-6 in EMT and peritoneal membrane dysfunction in long-term PD patients by assessing the level of IL-6 in dialysate and exploring the relationship between IL-6, the related signaling pathway JAK2/STAT3, and EMT, using in vitro cellular and molecular techniques. Plasma and dialysate levels of IL-6 were significantly higher in PD ultrafiltration failure patients compared with patients without ultrafiltration failure and were negatively correlated with measures of PD adequacy. In vitro IL-6 treatment changed human peritoneal mesothelial cell phenotype from a typical cobblestone-like to a fibroblast-like appearance and increased cell viability. IL-6 treatment increased α-smooth muscle actin and vascular endothelial growth factor expression but decreased E-cadherin expression. IL-6 treatment activated the JAK/STAT signaling pathway. However, the JAK2/STAT3 inhibitor WP1066 prevented IL-6-induced activation of the JAK2/STAT3 pathway and EMT. We conclude that IL-6 promotes the EMT process, possibly by activating the JAK2/STAT3 signaling pathway. IL-6 may serve as a novel therapeutic target for preventing EMT, and preservation of the peritoneal membrane may arise from these studies.

Caveolin-1 deficiency induces a MEK-ERK1/2-Snail-1-dependent epithelial-mesenchymal transition and fibrosis during peritoneal dialysis.

Peritoneal dialysis (PD) is a form of renal replacement therapy whose repeated use can alter dialytic function through induction of epithelial-mesenchymal transition (EMT) and fibrosis, eventually leading to PD discontinuation. The peritoneum from Cav1-/- mice showed increased EMT, thickness, and fibrosis. Exposure of Cav1-/- mice to PD fluids further increased peritoneal membrane thickness, altered permeability, and increased the number of FSP-1/cytokeratin-positive cells invading the sub-mesothelial stroma. High-throughput quantitative proteomics revealed increased abundance of collagens, FN, and laminin, as well as proteins related to TGF-ß activity in matrices derived from Cav1-/- cells. Lack of Cav1 was associated with hyperactivation of a MEK-ERK1/2-Snail-1 pathway that regulated the Smad2-3/Smad1-5-8 balance. Pharmacological blockade of MEK rescued E-cadherin and ZO-1 inter-cellular junction localization, reduced fibrosis, and restored peritoneal function in Cav1-/- mice. Moreover, treatment of human PD-patient-derived MCs with drugs increasing Cav1 levels, as well as ectopic Cav1 expression, induced re-acquisition of epithelial features. This study demonstrates a pivotal role of Cav1 in the balance of epithelial versus mesenchymal state and suggests targets for the prevention of fibrosis during PD.

Deficiency of endothelial nitric oxide signaling pathway exacerbates peritoneal fibrosis in mice.

BACKGROUND: Long-term peritoneal dialysis (PD) causes peritoneal dysfunction and structural alterations, eventually leading to peritoneal fibrosis. The endothelial nitric oxide synthase (eNOS)-NO signaling pathway contributes to the progression of organ fibrosis. However, it remains unknown whether NO signaling is involved in the process of peritoneal fibrosis. We evaluated the role of the eNOS-NO signaling pathway in the development of peritoneal fibrosis and whether stimulation of soluble guanylate cyclase (sGC), a downstream effector of NO, could attenuate peritoneal fibrosis. METHODS: We used wild-type (WT) and eNOS-deficient mice (eNOSKO). The mice underwent mechanical peritoneal stripping-induced peritoneal fibrosis at day 0. At 3, 7, 14, and 28 days after peritoneal stripping, the mice were killed. In some eNOSKO mice, the sGC stimulator Bay 41-2272 was administered by intraperitoneal injection. RESULTS: In WT mice, granulomatous tissue formation was observed in the submesothelial area at days 3 and 7. After day 7, the peritoneal membrane thickness gradually decreased and peritoneal tissue was repaired with leaving only slight fibrosis at day 28. However, eNOSKO mice demonstrated more progression of peritoneal fibrosis than WT mice at 28 days after peritoneal stripping. Expression of vimentin in the thickened peritoneum was prolonged after day 7 in eNOSKO mice. Treatment with Bay 41-2272 significantly attenuated peritoneal vimentin expression and fibrosis in the eNOSKO mice. CONCLUSIONS: Disruption of the eNOS-NO signaling pathway exacerbates peritoneal fibrosis by delaying wound healing. sGC stimulation may be a useful therapy for prevention of peritoneal fibrosis.

Activation of p38 mitogen-activated protein kinase promotes peritoneal fibrosis by regulating fibrocytes.

BACKGROUND: Peritoneal fibrosis is a serious complication of long-term peritoneal dialysis, and yet the precise pathogenic mechanisms of peritoneal fibrosis remain unknown. Fibrocytes participate in tissue fibrosis and express chemokine receptors that are necessary for migration. The p38 mitogen-activated protein kinase (MAPK) pathway regulates the production of chemokines and has been demonstrated to contribute to the pathogenesis of various fibrotic conditions. Accordingly, we used an experimental mouse model of peritoneal fibrosis to examine the dependency of fibrocytes on p38MAPK signaling. METHODS: Peritoneal fibrosis was induced in mice by the injection of 0.1% chlorhexidine gluconate (CG) into the abdominal cavity. Mice were treated with FR167653, a specific inhibitor of p38MAPK, and immunohistochemical studies were performed to detect fibrocytes and cells positive for phosphorylated p38MAPK. The involvement of p38MAPK in the activation of fibrocytes also was also investigated in vitro. RESULTS: Fibrocytes infiltrated peritoneum in response to CG, and that response was accompanied by progressive peritoneal fibrosis. The phosphorylation of p38MAPK, as defined by CD45+ spindle-shaped cells, was detected both in peritoneal mesothelial cells and in fibrocytes. The level of peritoneal expression of CCL2, a chemoattractant for fibrocytes, was upregulated by CG injection, and treatment with FR167653 reduced the number of cells positive for phosphorylated p38MAPK, the peritoneal expression of CCL2, and the extent of peritoneal fibrosis. Pretreatment with FR167653 inhibited the expression of procollagen type I α1 induced by transforming growth factor-ß1. CONCLUSIONS: Our results suggest that p38MAPK signaling contributes to peritoneal fibrosis by regulating fibrocyte function.

[Protein gene product 9.5-immunoactive nerve fibers and its clinical significance in endometriotic peritoneal lesions].

OBJECTIVE: To investigate the association between distribution of protein gene product (PGP) 9.5-immunoactive nerve fibers in peritoneal endometriotic lesions and disease-associated pain symptoms. METHODS: Thirty two peritoneal endometriotic lesions from patients with endometriosis (16 cases with pain and 16 cases without pain) and matched with 20 peritoneal tissues from patients with uterine leiomyoma without endometriosis were stained immunohistochemically for PGP9.5-immunoactive nerve fibers. RESULTS: The positive rate and density of PGP9.5-immunoreactive nerve fibers in peritoneal endometriotic leision were 62% (10/16) and (3.8+/-1.7)/mm2 in endometriosis patients with pain, which were significantly higher than 19% (3/16) and (1.7+/-0.5)/mm2 in endometriosis patients without pain (P<0.05) and 25% (5/20) and (1.3+/-0.6)/mm2 in peritoneal tissues in women without endometriosis (P<0.05). However, no differences were found between endometriosis patients without pain and women without endometriosis (P>0.05). Moreover, the density of PGP9.5-immunoreactive nerve fibers in peritoneal lesions in endometriosis patients with pain was positively correlated with the severity of pain (r=0.855, P<0.05). In addition, the density of PGP9.5-immunoreactive nerve fibers in peritoneal lesions was statistically higher in endometriosis patients with chronic pelvic pain and (or) dysmenorrhea than those in endometriosis patients with other type of pain (P<0.05), which was not associated with active lesion, site and staging (P>0.05). CONCLUSION: It suggested that PGP9.5-immunoreactive nerve fibers might confer the mechanism of pelvic pain with endometriosis.

GnRH receptor and peritoneal plasmin activity.

Most surgical procedures performed by obstetrician-gynecologists are associated with pelvic adhesions that cause subsequent serious sequelae, including small bowel obstruction, infertility, chronic pelvic pain, and difficulty in postoperative treatment, including complexity during subsequent surgical procedures. This study was conducted to determine if gonadotropin-releasing hormone analogues (GnRHa) affect the expressing tissue-type plasminogen activator (t-PA) and its inhibitor-1 (PAI-1) in peritoneal cells in culture. Human peritoneal Met5A cells were used to examine the effects of GnRHa leuprolide, buserelin and goserelin on the levels of t-PA and PA-1. Antigen concentrations were measured in conditioned media and cell lysates by real-time PCR and ELISA. GnRH receptor (GnRHR) mRNA was determined by RT-PCR. GnRHR mRNA was detected in Met5A cells. Exposure of Met5A cells to GnRHa induced a rapid decrease of PAI-1 level in cultured medium but not in cell lysate (protein and mRNA). These effects of GnRHa on PAI-1 were not associated with any changes in t-PA level. These results suggest that GnRHa may be an effective stimulator of local peritoneal fibrinolytic activity, as it decreases PAI-1 secretion in peritoneal Met5A cells by a mechanism linked to GnRHR.

Expression of eicosanoid biosynthetic and catabolic enzymes in peritoneal endometriosis.

BACKGROUND: Increased peritoneal eicosanoid concentrations have been reported in endometriosis patients and might be important in disease-associated pain and inflammation. Here, we evaluated the expression of key biosynthetic and catabolic enzymes involved in this abnormal eicosanoid production in peritoneal macrophages and endometriotic lesions. METHODS: Peritoneal macrophages, endometriotic lesions and matched eutopic endometrium were collected from endometriosis patients (n = 40). Peritoneal macrophages and eutopic endometrium samples were also collected from disease-free women (n = 25). Expression of type IIA secretory phospholipase A(2) (sPLA(2)-IIA), cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 5-lipoxygenase (5-LO) was quantified by real-time PCR, and these five key enzymes were localized by immunohistochemistry. RESULTS: sPLA(2)-IIA, COX-2 and mPGES-1 mRNA was significantly increased in peritoneal macrophages of endometriosis patients compared with controls (P = 0.006, P = 0.016 and P = 0.025, respectively). In endometriosis patients, sPLA(2)-IIA, mPGES-1 and 15-PGDH mRNA was significantly enhanced in peritoneal lesions compared with matched eutopic endometrium (P < 0.001, P < 0.001 and P = 0.005, respectively). In eutopic endometrium, a significant decrease in 15-PGDH mRNA was found in the endometriosis group compared with controls (P = 0.023). Finally, sPLA(2)-IIA, COX-2, mPGES-1 and 15-PGDH immunostaining was found mainly in endometrial glands, whereas 5-LO was distributed throughout the glands and stroma. CONCLUSIONS: Our study highlights an imbalance between eicosanoid biosynthesis and degradation in endometriosis patients. Both peritoneal macrophages and endometriotic lesions may be involved. Research into new molecules inhibiting biosynthetic enzymes (such as sPLA(2)-IIA and mPGES-1) and/or activating catabolic enzymes (such as 15-PGDH) may prove to be a major field of investigation in the development of targeted medical therapies.

Cilostazol and pentoxifylline decrease angiogenesis, inflammation, and fibrosis in sponge-induced intraperitoneal adhesion in mice.

AIMS: Adhesion formation following abdominal intervention is an abnormal peritoneal healing process. Our aim was to investigate the effects of controlling adhesion development by inhibiting its key components (angiogenesis, inflammation and fibrosis) using phosphodiesterase (PDE) inhibitors. MAIN METHODS: Two PDE inhibitors including cilostazol a PDE3 inhibitor (40 and 400 mg/kg), and pentoxifylline (PTX), a PDE 1-5 inhibitor (50 and 500 mg/kg) were used for a period of 7 days to inhibit angiogenesis, inflammation, and fibrosis in a murine model of sponge-induced peritoneal adhesion. Angiogenesis was assessed by hemoglobin content, vascular endothelial growth factor (VEGF) levels, and morphometric analysis. Accumulation of neutrophils and macrophages was determined by measuring myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities, respectively. Levels of TNF-alpha were also determined. Fibrosis was assessed by determining the amount of collagen in the implant; TGF-beta1 levels in the implant were also measured. KEY FINDINGS: Our results show that the treatments attenuated the main components of the adhesion tissue by reducing the amount of fibrovascular tissue that infiltrated the sponge matrix (wet weight). Hemoglobin content and VEGF levels were also decreased by approximately 40%. Neutrophil accumulation was unaffected by the compounds. However, NAG activity was reduced by pentoxifylline, but not by cilostazol. These compounds also decreased the levels of the pro-inflammatory and pro-fibrogenic cytokines TNF-alpha and TGF-beta1, respectively, and collagen synthesis. SIGNIFICANCE: Our results suggest that cilostazol and PTX decreased the development of peritoneal adhesions in the model, which might be associated with cyclic nucleotide modulation. Therapies to intervene in these pathways may be beneficial for the prevention of these lesions.

Cyclooxygenase-2 mediates dialysate-induced alterations of the peritoneal membrane.

During peritoneal dialysis (PD), exposure of the peritoneal membrane to nonphysiologic solutions causes inflammation, ultimately leading to altered structure and function. Myofibroblasts, one of the cell types that contribute to dysfunction of the peritoneal membrane, can originate from mesothelial cells (MCs) by epithelial-to-mesenchymal transition (EMT), a process that has been associated with an increased rate of peritoneal transport. Because cyclooxygenase-2 (COX-2) is induced by inflammation, we studied the role of COX-2 in the deterioration of the peritoneal membrane. We observed that nonepithelioid MCs found in peritoneal effluent expressed higher levels of COX-2 than epithelioid MCs. The mass transfer coefficient for creatinine correlated with MC phenotype and with COX-2 levels. Although COX-2 was upregulated during EMT of MCs in vitro, COX-2 inhibition did not prevent EMT. In a mouse model of PD, however, COX-2 inhibition with Celecoxib resulted in reduced fibrosis and in partial recovery of ultrafiltration, outcomes that were associated with a reduction of inflammatory cells. Furthermore, PD fluid with a low content of glucose degradation products did not induce EMT or COX-2; the peritoneal membranes of mice treated with this fluid showed less worsening than mice exposed to standard fluid. In conclusion, upregulation of COX-2 during EMT may mediate peritoneal inflammation, suggesting COX-2 inhibition as a potential strategy to ameliorate peritoneal deterioration in PD patients.

Vulnerability to oxidative stress and different patterns of senescence in human peritoneal mesothelial cell strains.

Both the ascites fluid-derived mesothelial cell line LP-9 and primary cultures of human omentum-derived mesothelial cells (HOMCs) are commonly used in experimental studies. However, they seem to have a different replicative potential in vitro. In the present study, we have attempted to determine the causes of this discrepancy. HOMCs were found to divide fewer times and enter senescence earlier than LP-9 cells. This effect was coupled with earlier increases in the expression of senescence-associated-beta-galactosidase and cell cycle inhibitors p16INK4a and p21WAF1. Moreover, almost 3 times as many early-passage HOMCs as LP-9 cells bore senescence-associated DNA damage foci. In sharp contrast to LP-9 cells, the foci present in HOMCs localized predominantly outside the telomeres, and the HOMC telomere length did not significantly shorten during senescence. Compared with LP-9 cells, HOMCs were found to enter senescence with significantly lower levels of lipofuscin and damaged DNA, and markedly decreased glutathione contents. In addition, early-passage HOMCs generated significantly more reactive oxygen species either spontaneously or in response to exogenous oxidants. These results indicate that compared with LP-9 cells, HOMCs undergo stress-induced telomere-independent premature senescence, which may result from increased vulnerability to oxidative DNA injury.

Hypoxia regulates iNOS expression in human normal peritoneal and adhesion fibroblasts through nuclear factor kappa B activation mechanism.

OBJECTIVE: To determine the mechanism by which hypoxia increases expression of iNOS in human normal peritoneal and adhesion fibroblasts. DESIGN: Prospective experimental study. SETTING: University medical center. PATIENT(S): Primary cultures of fibroblasts from normal peritoneum and adhesion tissues. INTERVENTION(S): Hypoxia-treated cells. MAIN OUTCOME MEASURE(S): We used real-time reverse transcription-polymerase chain reaction to quantify mRNA levels of iNOS and nuclear factor kappa B (NF-kappaB). Western blots were used to determine iNOS, NF-kappaB, IkappaB-alpha, and phospho-IkappaB expression levels in normal peritoneal and adhesion fibroblasts in response to hypoxia. RESULT(S): Hypoxia resulted in a significant increase in iNOS and NF-kappaB expression in normal and adhesion fibroblasts. Furthermore, both cell types manifested lower levels of NF-kappaB, cytoplasmic phospho-IkappaB-alpha, and iNOS proteins. In contrast, they manifested higher levels of cytoplasmic IkappaB-alpha and IkappaB-alpha/NF-kappaB ratios as well as a phosphorylated-IkappaB-alpha/NF-kappaB ratio. Under hypoxic conditions, both cell types exhibited significantly decreased cytoplasmic NF-kappaB, IkappaB-alpha levels, and significantly increased cytoplasmic phospho-IkappaB-alpha, iNOS, and NF-kappaB protein levels. CONCLUSION(S): Hypoxia increases iNOS expression by a mechanism involving activation of NF-kappaB. The ratio of IkappaB-alpha/NF-kappaB or IkappaB-alpha/p-IkappaB-alpha can be used to monitor activation.

O uso de telas Parietex® e Surgisis® na correção de defeitos produzidos na parede abdominal de coelhos / The repair of abdominal defects in rabbits with Parietex® and Surgisis® meshes abdominal wall

RACIONAL: Na cirurgia geral, as correções das hérnias da parede abdominal ocupam lugar de destaque e, cada vez mais, as indicações e usos de telas têm aumentado devido aos melhores resultados. OBJETIVO: Comparar as correções de orifícios produzidos em parede abdominal com telas Parietex® e Surgisis® em contato direto com as vísceras abdominais. MÉTODO: Para os experimentos foram utilizadas 16 coelhas adultas jovens e produção de defeitos triangulares de 2 cm de base por 2,5 cm de altura, comprometendo os planos músculo-aponeurótico-peritoniais da parede abdominal, nos flancos, simétricos à linha média que foram corrigidos com telas retangulares de 3 cm de base por 3,5 cm de altura. No lado direito usou-se tela Parietex® (poliéster/colágeno-polietilenoglicol-glicerol) e no lado esquerdo tela Surgisis® (submucosa intestinal suína). Na avaliação utilizaram-se parâmetros clínico-cirúrgicos, histológicos e imunoistoquímicos. Oito coelhas foram submetidas a eutanásia em 30 dias e as 8 restantes, em 60. Comparou-se a eficiência das duas telas. RESULTADOS: As duas telas provocaram erosões de pele e não ocorreu nenhum caso de hérnia incisional. As aderências ocorreram em todas as telas no primeiro mês e em menor grau e intensidade, no segundo mês; a retração delas foi de 1/3 do tamanho original; a Parietex® provocou menor processo inflamatório; não houve diferença significante de deposição de entre as duas telas; a deposição do colágeno tipo III foi mais intensa no segundo mês em ambas; na remodelação do colágeno a produção da enzima MMP8 foi maior na tela Parietex® no primeiro mês e a enzima MMP13 aumentou no segundo mês em ambas as telas, porém com significância apenas na Parietex®. CONCLUSÃO: As duas telas foram eficientes na correção de hérnias incisionais e com resultados semelhantes, sendo que a Parietex® apresentou menor processo inflamatório, maior quantidade de metaloproteinases MMP8 e MMP13 em relação à Surgisis®.

BACKGROUND: In general surgery, the repair of abdominal wall hernias has a prominent place, and the indications and uses of meshes have increased due to better results. AIM: To compare the repair of induced abdominal wall defects with Parietex® and Surgisis® meshes, in direct contact with abdominal viscera (intraperitoneal mesh). METHOD: For the experiments, were used 16 female young adult rabbits. Two full thickness triangular defects of 2 cm base by 2.5 cm high were created, lateral to the linea alba, one at each side. They were repaired with rectangular meshes of 3 cm base by 3.5 cm high, on the right side with Parietex® mesh (polyester/collagen-polyethylenglycol-glycerol), and on the left side with Surgisis® mesh (lyophilized porcine small bowel submucosa). The evaluation included clinical-surgical findings as well as histological and immunohistochemical parameters. Eight rabbits were subjected to euthanasia after 30 days, and the eight after 60 days. RESULTS: Both meshes induced skin erosions, despite the varying levels of mesh undermining evaluated, no incisional hernia occurred. There were peritoneal adhesions to the surface of both types of meshes after 30 days and in a lower extent and intensity after 60 days. Meshes' shrinking correspond to 1/3 of the original size and Parietex® caused less inflammatory process at the histologic evaluation. Deposition of collagen type I presented no significant difference between the meshes, but deposition of collagen type III was more intense after 60 days, in both groups. Regarding collagen's rearrangement, the production of MMP8 was higher on Parietex® after 30 days, and MMP13 enzyme was increased after 60 days, in both meshes (significant only for Parietex®). CONCLUSION: Both meshes were efficient in the correction of abdominal wall defects, and with similar results, but Parietex® presented less inflammatory process and greater amount of matrix-metalloproteinases MMP8 and MMP13 than Surgisis®.

Targeting a metalloprotease-PAR1 signaling system with cell-penetrating pepducins inhibits angiogenesis, ascites, and progression of ovarian cancer.

Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer.

[The repair of abdominal defects in rabbits with Parietex and Surgisis meshes abdominal wall]. / O uso de telas Parietex e Surgisis na correção de defeitos produzidos na parede abdominal de coelhos.

BACKGROUND: In general surgery, the repair of abdominal wall hernias has a prominent place, and the indications and uses of meshes have increased due to better results. AIM: To compare the repair of induced abdominal wall defects with Parietex and Surgisis meshes, in direct contact with abdominal viscera (intraperitoneal mesh). METHOD: For the experiments, were used 16 female young adult rabbits. Two full thickness triangular defects of 2 cm base by 2.5 cm high were created, lateral to the linea alba, one at each side. They were repaired with rectangular meshes of 3 cm base by 3.5 cm high, on the right side with Parietex mesh (polyester/collagen-polyethylenglycol-glycerol), and on the left side with Surgisis mesh (lyophilized porcine small bowel submucosa). The evaluation included clinical-surgical findings as well as histological and immunohistochemical parameters. Eight rabbits were subjected to euthanasia after 30 days, and the eight after 60 days. RESULTS: Both meshes induced skin erosions, despite the varying levels of mesh undermining evaluated, no incisional hernia occurred. There were peritoneal adhesions to the surface of both types of meshes after 30 days and in a lower extent and intensity after 60 days. Meshes' shrinking correspond to 1/3 of the original size and Parietex caused less inflammatory process at the histologic evaluation. Deposition of collagen type I presented no significant difference between the meshes, but deposition of collagen type III was more intense after 60 days, in both groups. Regarding collagen's rearrangement, the production of MMP8 was higher on Parietex after 30 days, and MMP13 enzyme was increased after 60 days, in both meshes (significant only for Parietex). CONCLUSION: Both meshes were efficient in the correction of abdominal wall defects, and with similar results, but Parietex presented less inflammatory process and greater amount of matrix-metalloproteinases MMP8 and MMP13 than Surgisis.

[Matrix metalloproteinase 9 presence in adhesive intraperitoneal processes]. / Studiul prezentei metaloproteinazei 9 la nivelul proceselor aderentiale cronice intraperitoneale.

UNLABELLED: Matrix metalloproteinases (MMP) represents a zinc-dependent proteolytic enzyme multigenic family, directly involved in embryogenesis, some physiological and pathological processes, occurring in the adult organism. For physiological processes, MMP play roles in proliferation, tissue remodeling, wound healing, angiogenesis. AIM: The aim of the study was to analyze the MMP9 presence in chronic peritoneal adherences, following the correlation between the presence of this metalloproteinase and the evolution of the connective tissue remodeling process from intraperitoneal adherences. MATERIAL AND METHODS: 6 women formed the study group: 5 operated for gynecological pathologies (with harvested tissue fragments from the adherences) and one patient with harvested tissue from normal peritoneum. Fragments were fixed in 10% formalin and processed for the microscopic exam in routine staining and immunohistochemistry, using anti-MMP9 antibodies and a technique based on peroxidaze-antiperoxidase system. The immunohistochemical reaction was quantified by using image analysis software. RESULTS: The morphological structure of the adherences was mainly represented by fibrous tissue, sometimes associated with elements of muscle and adipose tissues. For the fibrous connective tissue, the collagen component was characterized by a fascicular organization with different orientations and with fibrillar aspect. Semiquantitative analysis showed increased levels for MMP9 in adherences by comparison with normal peritoneum (mean ration for the MMP9-positive area: 26.5% for the adherences versus 15.93% normal peritoneum). CONCLUSION: The intraperitoneal adherences with more than 1 year of evolution show an extension of the remodeling processes that can lead to the possible resolution.

A neurokinin-1 receptor antagonist that reduces intra-abdominal adhesion formation decreases oxidative stress in the peritoneum.

Oxidative stress has been implicated in intra-abdominal adhesion formation. Substance P, a neurokinin-1 receptor (NK-1R) ligand, facilitates leukocyte recruitment and reactive oxygen species (ROS) generation. We have shown in a rat model of adhesion formation that intraperitoneal administration of a NK-1R antagonist at the time of abdominal operation reduces postoperative adhesion formation. Thus we determined the effects of NK-1R antagonist administration on peritoneal leukocyte recruitment and oxidative stress within 24 h of surgery. Adhesions were induced in Wistar rats randomly assigned to receive the antagonist or vehicle intraperitoneally. Peritoneal tissue was isolated at 2, 4, 6, and 24 h after surgery for analysis of the oxidative stress biomarkers 8-isoprostane (8-IP), protein carbonyl, NADPH oxidase, myeloperoxidase (MPO), and ICAM-1 and VCAM-1 mRNAs. Total antioxidant capacity of peritoneal fluid was also determined. MPO, NADPH oxidase, 8-IP, and protein carbonyl were elevated (P < 0.05) by 6 h. ICAM-1 mRNA was elevated (P < 0.05) by 2 h, whereas VCAM-1 levels decreased (P < 0.05) at 24 h. The NK-1R antagonist delayed the MPO rise and reduced (P < 0.05) 8-IP levels by 6 h and ICAM-1 mRNA, VCAM-1 mRNA, and protein carbonyl at 2 h. The antagonist also increased (P < 0.05) the antioxidant capacity of peritoneal fluid at all time points. These data further support a role for oxidative stress in adhesion formation and suggest that the NK-1R antagonist may limit adhesions, in part, by reducing postoperative oxidative stress through an inhibition of neutrophil recruitment and an increase in peritoneal fluid antioxidant capacity.

Peritoneal cell-derived mast cells: an in vitro model of mature serosal-type mouse mast cells.

Bone marrow-derived mast cells (BMMC) have been used extensively as a mast cell model. BMMC, however, are immature cells that have no known physiological equivalent in tissues. They do not respond to IgG immune complexes. They may therefore not be appropriate for studying the physiopathology of IgE-induced allergies or IgG-induced tissue-specific inflammatory diseases which both depend on mature mast cells. Resident peritoneal mast cells are a minor population of differentiated cells that are not readily purified. They, however, can be expanded in culture to generate large numbers of homogeneous cells. We show here that these peritoneal cell-derived mast cells (PCMC) are mature serosal-type mouse mast cells which retain most morphological, phenotypic, and functional features of peritoneal mast cells. Like peritoneal mast cells, PCMC respond to IgG Abs. IgG immune complex-induced responses depended on FcgammaRIIIA and were negatively regulated by FcgammaRIIB. We found that a moderate FcgammaRIIB-dependent negative regulation, due not to a higher FcgammaRIIIA/FcgammaRIIB ratio, but to a relatively inefficient use of the lipid phosphatase SHIP1, determines this property of PCMC. PCMC also respond to IgE Abs. IgE-induced PCMC responses, however, differed quantitatively and qualitatively from BMMC responses. PCMC secreted no or much lower amounts of lipid mediators, chemokines, and cytokines, but they contained and released much higher amounts of preformed granular mediators. PCMC, but not BMMC, also contained and, upon degranulation, released molecules with a potent proteolytic activity. These properties make PCMC a useful new model for understanding the physiopathology of mast cells in IgE- and IgG-dependent tissue inflammation.