Locally increased concentrations of inflammatory cytokines in an experimental intraabdominal adhesion model

Background

Peritoneal adhesions may cause bowel obstruction, infertility, and pain. This study investigated cytokines, proteins and growth factors thought to promote formation of adhesions in an experimental intraabdominal adhesion model.

Methods

Male Sprague-Dawley rats were subjected to laparotomy, cecal abrasion, and construction of a small bowel anastomosis and examined at various time points after surgery. Concentrations of cytokines and growth factors in plasma and peritoneal fluid were analyzed using electrochemoluminescence and quantitative sandwich enzyme immunoassay technique.

Results

Concentrations of interleukin-6 (IL-6), interleukin-1beta (IL-1β), and tumor necrosis factor alpha (TNF-α) increased in peritoneal fluid from 6 h after incision. Plasma concentrations of IL-6 increased at 6 h, but plasma concentrations of IL-1β and TNF-α remained low. Peritoneal fluid concentrations of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor beta1 (TGF-β1), vascular endothelial growth factor (VEGF), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were below detection levels at all time points.

Conclusion

Early elevations of IL-6, IL-1β, and TNF-α concentrations in peritoneal fluid correlated to adhesion formation in this rodent model. Our model is relevant and reproducible, suitable for intervention, and indicates that antiadhesion strategies should be early, local and not systemic.     

Smad7 inhibits TGF-β1-induced MCP-1 upregulation through a MAPK/p38 pathway in rat peritoneal mesothelial cells

Purpose

The TGF-β1/Smad signaling pathway is subject to inhibition by Smad7. High expression of Smad7 in the peritoneum of rats can delay and attenuate not only peritoneal fibrosis, but also monocyte infiltration into the peritoneum. The aim of this study was to investigate the anti-inflammatory mechanism of Smad7 in peritoneal fibrosis.

Methods

Rat peritoneal mesothelial cells were stimulated with TGF-β1, and the expression of MCP-1 protein and mRNA was measured. Furthermore, the expression of MCP-1 was determined following inhibition of TGF-β/Smad or p38 signaling using Smad7 transfection or SB203580 (10 μmol/L), respectively. The effect of exogenous Smad7 and SB203580 on activation of the TGF-β/Smad and p38 signaling pathways was also studied.

Results

TGF-β1 significantly upregulated the expression of MCP-1 at both the protein and mRNA level in a time-dependent manner. Exogenous Smad7 and SB203580 markedly inhibited TGF-β1-induced MCP-1 expression. Moreover, high expression of exogenous Smad7 not only inhibited phosphorylation of Smad2 and Smad3, but also diminished the level of phosphorylated p38. However, SB203580 had no effect on the phosphorylation of Smad2 and Smad3.

Conclusions

TGF-β1 exhibits pro-inflammatory effects through the upregulation of MCP-1 in peritoneal fibrosis. Smad7 inhibits TGF-β1 induced MCP-1 upregulation through a MAPK/p38-dependent pathway.     

An evaluation of normal saline and taurolidine on intra-abdominal adhesion formation and peritoneal fibrinolysis

BACKGROUND: Intraoperative lavage with normal saline or taurolidine solutions is commonly used in abdominal surgery. For this purpose, normal saline and taurolidine, which may modify the intrinsic fibrinolytic activity of the peritoneum by breaking the peritoneal adhesions, are frequently used. We aimed to evaluate how normal saline and taurolidine affect peritoneal fibrinolysis and adhesion formation. METHODS: A rat model of peritoneal adhesion was used. Control animals received no medication, while normal saline or taurolidine solutions were administered intraperitoneally to the remaining two groups (n = 20 for each group). At postoperative day 10 adhesions were graded, and cardinal parameters of peritoneal fibrinolysis (activities and concentrations of tissue plasminogen activator [tPA], plasminogen activator inhibitor type 1 [PAI-1], and tPA/PAI-1 complex), and hydroxyproline contents were determined in peritoneal tissue samples. RESULTS: Total adhesion scores were decreased in both of the treated groups. Median tissue levels of tPA and tPA activity were higher in the treated groups versus controls. The PAI-1 levels were similar among the three groups. tPA/PAI-1 complex levels were higher in animals that received normal saline and in those treated with taurolidine solution compared with control animals. Peritoneal hydroxyproline levels were similar across the three groups. CONCLUSIONS: Our results suggest that normal saline and taurolidine solution administrations might reduce peritoneal adhesion formation, probably by altering peritoneal fibrinolytic activity.     

Fibrinolytic responses of human peritoneal fluid in laparoscopic cholecystectomy: a prospective clinical study

Background The reduction in peritoneal fibrinolysis is believed to be the pathogenetic mechanism of adhesion formation. The general conclusion based on previous clinical and experimental studies is that laparoscopic procedures produce less adhesion formation. The association between this beneficial effect of laparoscopic cholecystectomy and peritoneal fibrinolytic changes is not clear. Therefore, the authors aimed to compare the effects of open and laparoscopic cholecystectomy on peritoneal fibrinolysis. For this purpose, fibrinolytic parameters in peritoneal fluid were investigated 24 h after laparoscopic and open cholecystectomies. Methods In a prospective clinical study, peritoneal fluid was sampled via a drain 24 h after laparoscopic (n = 10) and open (n = 9) cholecystectomies. Activities and concentrations of tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and tPA/PAI-1 complex were determined by enzyme-linked immunosorbent assay (ELISA) kits. Results In peritoneal fluids, tPA and tPA/PAI-1 complex concentrations were higher in the open cholecystectomy group (p = 0.009 and p < 0.001, respectively), but tPA activity and PAI-1 concentrations did not differ between the groups (p = 0.514 and p = 0.716, respectively). Conclusions Fibrinolytic changes in peritoneal fluid have several similarities in open and laparoscopic cholecystectomies with regard to tPA activity and PAI-1 levels. However, higher tPA levels after the open procedure probably are secondary to more intense tissue handling leading to mesothelial release of tPA.     

Reduction of adhesion formation by an angiotensin type 1 receptor antagonist

Purpose  

Adhesion formations are important causes of intestinal obstruction and can lead to infertility in women. The formation of adhesion appears to be determined by the fibrinolytic activity. Fibrinolysis itself is controlled by the plasminogen activator system, and several studies have shown that angiotensin type 1 (AT₁) receptor antagonists can reduce plasminogen activator inhibitor-1 (PAI-1) expression, but the effect of AT₁ receptor antagonists on PAI-1 expression involved in the adhesion formation remains unclear. The aim of this study was to investigate whether an AT₁ antagonist, candesartan, can reduce PAI-1 mRNA expression using experimental model of peritoneal adhesions, which seems to reflect post-operative adhesions.     

Investigation of the Antifibrotic Effect of IFN-γ on Fibroblasts in a Cell Culture Model of Peyronie's Disease

Objective

A broad spectrum of options is available for treatment of Peyronie's disease; however, the effects of minimally invasive therapy are generally inadequate. Although useful, oral drugs must be administered at onset of the disease. Only a few patients request penile surgery. Therefore, new medical treatments for Peyronie's disease are needed. A better understanding of the pathogenesis of Peyronie's disease is required to facilitate development of these new medical treatments. Several studies have described an increased level of TGF-β in the fibrotic plaques of patients with Peyronie's disease, underscoring this important signalling pathway in the onset and/or development of Peyronie's disease.

Methods

Plaque biopsies were taken from 16 patients with Peyronie's disease. Furthermore, 7 patients without Peyronie's disease were biopsied to provide control material. Fibroblasts were cultured from biopsy tissue, and cultured fibroblasts were stimulated with TGF-β1, BMP-2, IFN-γ, and IFN-γ combined with one of the other stimuli. Protein was extracted from treated fibroblasts and prepared for immunoblots. The membranes were probed for phosphorylated Smad and total Smad to indicate activation of TGF-β signalling.

Results

An agonistic effect of IFN-γ on TGF-β signalling was observed. Stimulation with TGF-β1 increased levels of phospho-Smad2 and phospho-Smad3. After stimulation with TGF-β1 and IFN-γ combined, the levels of phospho-Smads were higher than those observed with stimulation withTGF-β1 alone.

Conclusions

The profibrotic effect of TGF-β1 is enhanced by IFN-γ in fibroblasts from patients with Peyronie's disease. The inhibitory effects of IFN-γ on the TGF-β pathway do not appear in Peyronie's disease. Therefore, IFN-γ cannot be taken as a useful tool in the therapy of Peyronie's disease.     

Stimulation of Protracted Connective Tissue Repair in Normal Mice by Transforming Growth Factorβ

The repair and contraction during connective tissue repair of mesenteric perforations is prolonged in mice compared with rats. In the present study the stimulating effect of transforming growth factor β₁, (TGF-β₁) on different aspects of such repair of the mouse mesentery was assessed. The number of closed mesenteric perforations were counted on different days after operation and the free peritoneal cells were counted, the mitotic index was assessed, and actin distribution of fibroblasts around the perforation was studied with laser scanning confocal microscopy. TGF-β₁ significantly increased the speed of closure and seemed to induce more actin in fibroblasts at the wound margin. It did not significantly influence the mitotic index, but fewer free peritoneal cells were obtained in mice treated with TGF-β₁. We conclude that TGF-β₁ is a potent stimulator of connective tissue repair and contraction in mice. The different methods of closure in rats and mice implicate different molecular responses in wounds and further studies on the stimulating effect of TGF-β₁ may indicate basic fibroblastic cellular mechanisms that are active during contraction in connective tissue repair.     

Smad4小分子干扰RNA对转化生长因子-β诱导的纤溶酶原激活物抑制剂-1表达的影响

目的 探讨Smad4小分子干扰RNA(siRNA)对转化生长因子(TGF)-β诱导纤溶酶原激活物抑制剂(PAI)-1的表达的影响及作用机制.方法 将Smad4 siRNA转染入肝星状细胞24h后行TGF-β(1μg/L)刺激,24、48 h后收集细胞.利用萤光素酶报告基因法测定Smad结合转写因子的活性;Northern印迹分析技术检测PAI-1 mRNA表达的变化;Western印迹分析观察Smad4蛋白、PAI-1蛋白的变化.结果 Smad4 siRNA能抑制Smad4蛋白(3.0±0.1比16.0±0.5)表达;Smad4 siRNA抑制TGF-β1和ALK5激活的4×SBE荧光素酶报告基因活性;TGF-β时间依赖性促进PAI-1 mRNA表达,24 h达高峰;Smad4 siRNA从mRNA(1.3±0.1比6.5±0.2)和蛋白(1.50±0.15比5.52±0.36)水平抑制TGF-β激活的PAL-1的表达.结论 Smad4 siRNA能够有效地阻断TGF-β诱导的PAI-1表达.     

Effects of seprafilm on peritoneal fibrinolytic system

BACKGROUND: There is a high incidence of adhesions after ventral hernia repair with polypropylene mesh. The purpose of the present study was to evaluate the efficacy of Seprafilm in the prevention of adhesion formation and effect on peritoneal fibrinolytic activity. METHODS: An incisional hernia model was created in rats. In the experimental group Seprafilm was placed between polypropylene mesh and abdominal organs. On the 14th day adhesions were evaluated and tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI) type 1 and 2 were measured in peritoneal biopsy specimens. Results: Adhesions were significantly reduced in the Seprafilm group (P = 0.002). Nevertheless, there were no difference between the two groups in levels of tPA, PAI-1 and PAI-2. However, the levels of uPA were significantly decreased in the Seprafilm group. CONCLUSIONS: The adhesion preventive effect of Seprafilm is not directly related in peritoneal fibrinolytic activity. Instead, the physical properties (barrier, hydroflotation and sliconizing effect) of the membrane are primarily responsible for adhesion prevention.     

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

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

The neurokinin 1 receptor regulates peritoneal fibrinolytic activity and postoperative adhesion formation

Background

Intra-abdominal adhesions are a common source of postoperative morbidity. Previous studies in our laboratory have shown that a neurokinin 1 receptor antagonist (NK-1RA) reduces abdominal adhesion formation and increases peritoneal fibrinolytic activity. However, the cellular pathway by which the antagonist exerts its effects is unclear, as cultured peritoneal mesothelial cells exposed to the NK-1RA show increases in fibrinolytic activity despite having very low expression of neurokinin 1 receptor (NK-1R) messenger RNA and protein. Our aim was to determine whether the NK-1R plays an essential role in the adhesion-reducing effects of the NK-1RA, or if the NK-1RA is acting independently of the receptor.

Methods

Homozygous NK-1R knockout mice and age matched wild-type mice underwent laparotomy with cecal cautery to induce adhesions. At the time of surgery, mice received a single intraperitoneal dose of either NK-1RA (25 mg/kg) or saline alone. Adhesion severity at the site of cecal cautery was assessed on postoperative day 7. In a separate experiment, peritoneal fluid was collected from wild type and NK-1R knockout mice 24 h after laparotomy with cecal cautery and administration of either NK-1RA or saline. Tissue plasminogen activator levels, representative of total fibrinolytic activity, were then measured in peritoneal fluid.

Results

In wild-type mice, NK-1RA administration significantly decreased adhesion formation compared with saline controls. Among the NK-1R knockout mice, there was no significant reduction in adhesion formation by the NK-1RA. Fibrinolytic activity increased 244% in wild-type mice administered NK-1RA compared with saline controls; however, the NK-1RA did not raise fibrinolytic activity above saline controls in NK-1R knockout mice.

Conclusions

These data indicate that the NK-1R mediates the adhesion-reducing effects of the NK-1RA, in part, by the upregulation of peritoneal fibrinolysis, and suggest that the NK-1R is a promising therapeutic target for adhesion prevention.     

TGF-β1 Inhibits Cx43 Expression and Formation of Functional Syncytia in Cultured Smooth Muscle Cells from Human Detrusor

Background

Human detrusor smooth muscle cells (hBSMCs) are coupled by connexin 43 (Cx43)–positive gap junctions to form functional syncytia. Gap junctional communication likely is necessary for synchronised detrusor contractions and is supposed to be altered in voiding disturbances. Other authors have shown that the pleiotropic cytokine TGF-β1 upregulates Cx43 expression in human aortic smooth muscle cells.

Objective

In this study, we examined the TGF-β1 effects on Cx43 expression in cultured hBSMCs.

Design, Setting, and Participants

hBSMC cultures, established from patients undergoing cystectomy, were treated with recombinant human TGF-β1.

Measurements

Cx43 expression was then examined by Western blotting, real-time PCR, and immunocytochemistry. Dye-injection experiments were used to study the size of functional syncytia.

Results and Limitations

Dye-coupling experiments revealed stable formation of functional syncytia in passaged cell cultures (P1–P4). Stimulation with TGF-β1 led to significant reduction of Cx43 immunoreactivity and coupling. Cx43 protein expression was significantly downregulated and Cx43 mRNA was only 30% of the control level. Interestingly, low phosphorylation species of Cx43 were particularly affected.

Conclusions

Our experiments demonstrated a significant down regulation of connexin 43 by TGF-β1 in cultured hBSMCs. These findings support the view that TGF-β1 is involved in the pathophysiology of urinary bladder dysfunction.     

Study of Transforming Growth Factor-β1 Gene,mRNA, and Protein in Japanese Renal Transplant Recipients

Background

Transforming growth factor (TGF)-β1 may contribute to chronic allograft nephropathy and graft loss; however, the exact molecular mechanism remains unclear. Therefore, we assess the relationship between TGF-β1 gene polymorphisms, expression, and development of allograft nephropathy.

Methods

We studied 135 renal transplant recipients at our hospital. TGF-β1 gene polymorphisms (codons 10 and 25) were determined from peripheral blood leukocyte DNA. Plasma TGF-β1 mRNA was measured by real-time polymerase chain reaction and TGF-β1 protein levels were assessed by enzyme-linked immunosorbent assay. The relationship between TGF-β1 genotyping, expression, and rejection and results of renal biopsy were evaluated.

Results

The genotype frequency of transplant recipients was 49.6%, 30.4%, and 20.0% for C/T, C/C and T/T at codon 10, 100% for G/G at codon 25, respectively. According to the criteria of Banff ‘97 classification, 24 cases were classified as acute rejection and whose genotypes were 16, 3, and 5 cases for C/T, C/C and T/T at codon 10. Plasma mRNA expression was elevated in 14 cases and decreased in 8 cases after acute rejection. We measured 267 specimens of TGF-β1 protein and there was no relation between amount of TGF-β1 protein and mRNA.

Conclusion

Our results suggest that the relationship between plasma TGF-β1 expression and the development of allograft nephropathy remains uncertain. Frequency of allograft rejection differ with TGF-β1 codon 10 genotypes and the high-risk genotype was different from the reports of other countries.     

Urinary Transforming Growth Factor-β1 Excretion in Renal Allograft Recipients During the Early Post-transplantation Period

Background.?Transforming growth factor-β₁ (TGF-β₁), the major fibrogenic growth factor, is implicated in the pathogenesis of renal scarring in experimental and clinical nephropathies as well as in chronic allograft nephropathy. In this study we examined the pattern of changes of TGF-β₁ excretion in the urine and the sites of TGF-β₁ expression in the kidney of transplanted patients during the early post-transplantation period. Methods.?Eighteen renal allograft recipients were included in the study. In all patients urinary TGF-β₁ levels were determined by ELISA in sequential measurements during the first two postoperative months and compared to that of 14 healthy subjects. The renal expression of TGF-β₁ protein was studied in 4 patients that underwent a biopsy of the transplanted kidney at the same period. All patients were treated with prednisolone, cyclosporin, and mycophenolate mofetil. Results.?Urinary TGF-β₁ levels were increased during the first postoperative days. Although they were gradually reduced during the first two post-operative months, they remained significantly higher compared to those of normal subjects (580 ± 148 ng/24 h vs. 310 ± 140 ng/24 h p<0.01). The decline of urinary TGF-β₁ excretion followed that of serum creatinine. TGF-β₁ protein expression was identified within the cytoplasm of tubular epithelial cells of transplanted patients. Conclusions.?Elevated urinary TGF-β₁ levels are observed during the early post-transplantation period in renal allograft recipients and are maintained high even after restoration of renal function to normal.     

Strong Smad4 Expression Correlates with Poor Prognosis After Surgery in Patients with Hepatocellular Carcinoma

Background

As a pleiotropic cytokine, transforming growth factor-β (TGF-β) controls the functions of proliferation, adhesion, and differentiation, and contributes to cancer promotion and suppression. Moreover, it is related to the epithelial–mesenchymal transition process and T cell differentiation associated with inflammation. The Smad4 protein is the downstream mediator of TGF-β. In this study, we examined the relationship between Smad4 expression and clinicopathological features in patients with hepatocellular carcinoma (HCC).

Methods

Expression of Smad4 was assessed in five HCC cell lines and in paired cancerous and noncancerous tissues in three patients with HCC, using Western blotting analysis. Moreover, Smad4 expression in 121 HCC patients was evaluated by using immunohistochemistry.

Results

Only the Li7 and HT17 cell lines expressed the Smad4 protein. All human samples expressed the protein. Immunohistochemistry showed that Smad4 expression tended to be strong in small HCC nodules less than 45 mm in diameter (P = 0.06) and in the infiltrated part of the tumor capsule. Postoperative survival analysis indicated that HCC patients with strong Smad4 expression had shorter disease-specific survival than those with weak expression (P = 0.04). Multivariate analysis also showed that Smad4 expression could be one predictor of prognosis, but the correlation was not significant (P = 0.07).

Conclusions

Although TGF-β/Smad4 signaling may have various biological effects on human malignancies, strong Smad4 expression in HCC is likely to suggest poor prognosis. The information has implications for predicting HCC prognosis and developing targeted therapeutics.