Human Peritoneal Fibroblasts Research Articles

Unravelling the role of Sildenafil and SB204741 in suppressing fibrotic potential of peritoneal fibroblasts obtained from PD patients.

Introduction: Peritoneal fibrosis (PF) results in technique failure in peritoneal dialysis (PD) patients. Peritoneal fibroblasts are characterized by increase in the ACTA2 gene, responsible for alpha smooth muscle actin (α-SΜΑ), extracellular matrix (ECM) production, and inflammatory cytokines production, which are the are key mediators in the pathogenesis of PF. 5-hydroxytryptamine (5-HT; serotonin) induces ECM synthesis in fibroblasts in a transforming growth factor-beta 1 (TGF-β1) dependent manner. The purpose of our study was to identify the potential mechanism and role of sildenafil and 5HT2B receptor inhibitor (SB204741) combination in attenuating PD-associated peritoneal fibrosis. Methods: Studies were performed to determine the effect of TGF-β1, sildenafil, and SB204741 on human peritoneal fibroblasts (HPFBs) isolated from the parietal peritoneum of patients in long-term PD patients (n = 6) and controls (n = 6). HPFBs were incubated with TGF-β1 (10ng/mL) for 1h and later with TGF-β1 (10ng/mL)/[sildenafil (10µM) or SB204741 (1µM)] and their combination for 24h (post-treatment strategy). In the pre-treatment strategy, HPFBs were pre-treated with sildenafil (10µM) or SB204741 (1µM) and a combination of the two for 1h and later with only TGF-β1 (10ng/mL) for 24h. Results: The anti-fibrotic effects of the combination of sildenafil and SB204741 were greater than that of each drug alone. In TGF-β1-stimulated HPFBs, pro-fibrotic genes (COL1A1, COL1A2, ACTA2, CTGF, FN1, and TGFB1) exhibited higher expression than in controls, which are crucial targets of sildenafil and SB204741 against peritoneal fibrosis. The synergistic approach played an anti-fibrotic role by regulating the pro- and anti-fibrotic gene responses as well as inflammatory cytokine responses. The combination treatment significantly attenuated peritoneal fibrosis, as evident by the almost complete amelioration of ACTA2 expression, restoration of anti-fibrotic genes (MMP2/TIMP1), and, at least, by reducing the expression of pro-inflammatory cytokines (IFN-γ, IL-4, IL-17, IL-1β, IL-6, TNF-α, and TGF-β1) along with an increase in IL-10 levels. Discussion: Taken together, the above research evidences that the combination of sildenafil and SB204741 may have therapeutic potential in suppressing peritoneal fibrosis due to peritoneal dialysis.

High Salt Promotes Inflammatory and Fibrotic Response in Peritoneal Cells.

Recent studies draw attention to how excessive salt (NaCl) intake induces fibrotic alterations in the peritoneum through sodium accumulation and osmotic events. The aim of our study was to better understand the underlying mechanisms. The effects of additional NaCl were investigated on human primary mesothelial cells (HPMC), human primary peritoneal fibroblasts (HPF), endothelial cells (HUVEC), immune cells (PBMC), as well as ex vivo on peritoneal tissue samples. Our results showed that a high-salt environment and the consequently increased osmolarity increase the production of inflammatory cytokines, profibrotic growth factors, and components of the renin-angiotensin-aldosterone system, including IL1B, IL6, MCP1, TGFB1, PDGFB, CTGF, Renin and Ace both in vitro and ex vivo. We also demonstrated that high salt induces mesenchymal transition by decreasing the expression of epithelial marker CDH1 and increasing the expression of mesenchymal marker ACTA2 and SNAIL1 in HPMCs, HUVECs and peritoneal samples. Furthermore, high salt increased extracellular matrix production in HPFs. We demonstrated that excess Na+ and the consequently increased osmolarity induce a comprehensive profibrotic response in the peritoneal cells, thereby facilitating the development of peritoneal fibrosis.

Cell Type-Specific Anti-Adhesion Properties of Peritoneal Cell Treatment with Plasma-Activated Media (PAM)

Postoperative abdominal adhesions are responsible for serious clinical disorders. Administration of plasma-activated media (PAM) to cell type-specific modulated proliferation and protein biosynthesis is a promising therapeutic strategy to prevent pathological cell responses in the context of wound healing disorders. We analyzed PAM as a therapeutic option based on cell type-specific anti-adhesive responses. Primary human peritoneal fibroblasts and mesothelial cells were isolated, characterized and exposed to different PAM dosages. Cell type-specific PAM effects on different cell components were identified by contact- and marker-independent Raman imaging, followed by thorough validation by specific molecular biological methods. The investigation revealed cell type-specific molecular responses after PAM treatment, including significant cell growth retardation in peritoneal fibroblasts due to transient DNA damage, cell cycle arrest and apoptosis. We identified a therapeutic dose window wherein specifically pro-adhesive peritoneal fibroblasts were targeted, whereas peritoneal mesothelial cells retained their anti-adhesive potential of epithelial wound closure. Finally, we demonstrate that PAM treatment of peritoneal fibroblasts reduced the expression and secretion of pro-adhesive cytokines and extracellular matrix proteins. Altogether, we provide insights into biochemical PAM mechanisms which lead to cell type-specific pro-therapeutic cell responses. This may open the door for the prevention of pro-adhesive clinical disorders.

Indigenous primary culture protocols for human adult skin fibroblast, pancreatic stellate cells, and peritoneal fibroblasts

Despite availability of commercially available cell lines, primary cultures of cells remain the best available natural resource for in vitro research work. However, establishing primary cultures is challenging and despite availability of many protocols for primary cell cultures reproducibility is lacking either due to cumbersome protocol, need for special reagents, and requirement of training. Herein, we present our simplistic least manipulative protocols for establishing primary human dermal fibroblasts, human pancreatic stellate cells, and human peritoneal fibroblasts that may be followed by other research laboratories for in vitro studies.

Talcum powder induces malignant transformation of human primary normal ovarian epithelial cells but not human primary normal peritoneal fibroblasts

Objective: The aim of this study was to demonstrate whether exposure to talcum powder induces malignant transformation in human primary normal ovarian epithelial cells and human primary normal peritoneal fibroblasts. Epidemiological and molecular studies have linked perineal use of talcum powder to increased risk of ovarian cancer. Exposure to talcum powder was shown to induce specific point mutations in key redox enzymes that altered their activities in both normal and epithelial ovarian cancer cells.

5‐HT2 and 5‐HT2B Receptor Antagonism Regulates Fibrotic Potential of Peritoneal Fibroblasts in CAPD by Targeting TGF‐β1‐Src‐ERK1/2‐STAT3 Pathway

Peritoneal fibrosis (PF) results in ultrafiltration failure in continuous ambulatory peritoneal dialysis (CAPD) patients. 5‐hydroxytryptamine (5‐HT; serotonin) induces extracellular matrix (ECM) synthesis in peritoneal fibroblasts in a transforming growth factor beta 1 (TGF‐β1) dependent manner. We evaluated anti‐fibrotic role of inhibitors of 5‐HT2 (Terguride) and 5‐HT2B (SB204741) in human peritoneal fibroblasts (HPFB) of CAPD patients. Peritoneal fibroblasts isolated fromCAPD patients (n=6) and controls (n=8), were incubated with 5‐HT (1μM)/TGF‐β1 (10ng/ml) for 1 hour and later with 5‐HT (1μM)/TGF‐β1 (10ng/ml) and terguride or SB204741 (1μM, each) for 24 hours (Post‐treatment strategy). In pre‐treatment strategy, cells were pre‐treated with terguride or SB204741 (1μM, each) for 1 hour and later with only 5‐HT (1μM)/TGF‐β1 (10ng/ml) for 24 hours. Real time PCR for pro‐fibrotic TGFB1, Type 1 collagen (COL1A1), Type 2 collagen (COL1A2), Actin alpha 2 (ACTA2), Connective tissue Growth factor (CTGF) and Fibronectin 1 (FN1) and anti‐fibrotic genes Matrix mettaloproteinase‐2/Tissue inhibitor of metalloproteinase (MMP2/TIMP1) expression was performed. Type I collagen and α‐SMA, phosphorylation status of Smad‐3, ERK1/2, Src and STAT‐3 was examined by western blotting.In 5‐HT/TGF‐β1stimulated HPFB, upregulated pro‐fibrotic gene expressionwas observed, which significantly reduced on co‐culture with 5‐HT2/5‐HT2B inhibitors, with no effect on anti‐fibrotic genes mRNA expression. In 5‐HT stimulated HPFB, treatment with both 5‐HT inhibitors decreased type 1 collagen and α‐SMA with reduced ERK1/2 phosphorylation (figure 1), however, Smad‐3 phosphorylation remain unaltered (figure 2). In 5‐HT/TGF‐β1 simulated HPFB, 5‐HT inhibitors decreased STAT3 phosphorylation, without affecting Src phosphorylation. 5‐HT receptor antagonists attenuate 5‐HT/TGF‐β1 mediated pro‐fibrotic potential of HPFB by suppressingTGF‐β1‐Src‐ERK1/2‐STAT3 pathway.Support or Funding InformationThis project received funding from intramural grant(Grant No ‐ 2017‐1‐IMP‐95) of Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.

Abstract AP20: STROMAL CELL EXPRESSION OF THE RECEPTOR TYROSINE KINASE DDR2 PROMOTES OVARIAN CANCER METASTASIS

Abstract OBJECTIVES: Understand the role of stromal discoid domain receptor 2 (DDR2) expression in ovarian cancer metastasis METHODS: 111 high-grade serous ovarian cancer specimens were examined for stromal DDR2 protein expression by immunohistochemistry (IHC) and correlated with survival. The stromal cell contribution of DDR2 for the steps of metastasis were evaluated through mesothelial cell clearance, Matrigel invasion assays, and germline knock-out mice. DDR2 low versus DDR2 high primary stromal cells were cultured from normal omenutum: human peritoneal mesothelial cells (HPMC) and normal omental fibroblasts (NOF). DDR2 knock-out (KO) and wild-type (WT) mice were used to evaluate metastasis. DDR2 positive (ID8Trp53-/-BRCA2-/- and ES2) tumor cells were used. Mesothelial cell clearance area was evaluated by measuring the size of the clearance area, invasion was evaluated by the number of tumor cells per high power field (hpf), and metastatic spread evaluated by number of tumors nodules and tumor weight. Collagen contribution was evaluated with Masson's trichrome stain and second harmonic generation (SHG). In addition, the media of genetically inhibited DDR2 or therapeutic inhibition of DDR2 in the NOFs (siControl vs siDDR2) was removed and then added to tumor cells to evaluate for invasion. Therapeutic inhibition was performed with a small molecule developed in a collaborating lab, WRG-R28. RESULTS: Patients with high stromal cell DDR2 expression had a median overall survival (OS) of 171 months vs. low stromal cell DDR2 expression (OS) of 28 months (p<0.0001). Mesothelial cell clearance was performed with HPMCsiControl vs siDDR2 and found to have decreased tumor cell clearance with HPMCsiDDR2 when compared to HPMCsiControl. Additionally, tumor cell invasion was decreased by 2-fold in the NOFsiDDR2 compared to NOFsiControl in two discrete NOFs. Furthermore, media from NOFsiControl vs NOFsiDDR2 was removed and co-cultured with DDR2+ tumor cells and found to have 50% less invasion for tumor cells co-cultured with NOFsiDDR2 compared to NOFsiControl. DDR2 KO mice had significantly less tumor weight (0.025g vs 0.05, p<0.05) than DDR2 WT mice. Collagen content was found to be less intense at 45% compared to 80%, p<0.0001 in the DDR2KO vs DDR2WT. SHG analysis showed DDR2 WT tumors to be more perpendicular than DDR2 KO tumors were more parallel to the tumor-stromal border. Therapeutic inhibition with WRG-R28 treatment of the NOFs resulted in 2-fold decrease in invasive tumor cells (150 vs 75 cells/hpf, p<0.01) as well as inhibition of mesothelial cell clearance by the tumor cells treated with WRG-R28 compared to DMSO. CONCLUSIONS: The stromal contribution of DDR2 promotes tumor cell clearance of mesothelial cells and metastatic spread. This suggests that stromal expression of DDR2 may be a potential target to guide future therapy particularly in the maintenance setting. Citation Format: Molly Greenwade, Holly Beck, Whitney Grither, Daniel Wilke, Katina Massad, Lei Guo, Greg Longmore, Katherine Fuh. STROMAL CELL EXPRESSION OF THE RECEPTOR TYROSINE KINASE DDR2 PROMOTES OVARIAN CANCER METASTASIS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP20.

MON-212 COMPLETE ABROGATION OF ALPHA SMOOTH MUSCLE ACTIN ON DUAL INHIBITION OF PHOSPHODIESTERASE 5 AND 5-HT2B INHIBITORS IN HUMAN PERITONEAL FIBROBLASTS ISOLATED FROM CAPD PATIENTS

Peritoneal fibrosis is a major cause of ultrafiltration failure in continuous ambulatory peritoneal dialysis (CAPD) patients. Transforming growth factor beta 1 (TGF-β1) activates resident fibroblasts which trans-differentiate into myofibroblasts (MFBs), characterized by increased alpha-smooth muscle actin (α-SMA) expression and extracellular matrix (ECM) proteins production. Our objective was to evaluate anti-fibrotic efficacy on dual inhibition of Phosphodiesterase 5 (PDE5) inhibitors, Sildenafil & Zaprinast, plus 5-HT2B inhibitor, SB204741, respectively in human peritoneal fibroblasts (HPFB). Biopsy from parietal peritoneum (PB) of control patients (n=8) and CAPD patients (n=6) excised during laparotomy was incubated overnight in dispase (2.4 U/mL)/37°C. In disease mimicking strategy, HPFB were incubated with TGF-β1 (10 ng/ml)/1 hr, later with TGF-β1 (10 ng/ml)/[Sildenafil (10 µM) plus SB204741 (1 µM) or Zaprinast (10 µM) plus SB204741 (1 µM)]/24 hr. In pre-treatment strategy, HPFB were pretreated with [Sildenafil (10 µM) plus SB204741 (1 µM) or Zaprinast (10 µM) plus SB20474 (1 µM)]/1 hr and later with only TGF-β1 (10 ng/ml)/24 hr. Similar strategies were followed for individual treatments of inhibitors. Real-time qPCR for pro-fibrotic (COL1A1, COL1A2, ACTA2, CTGF and FN1) and anti-fibrotic genes (MMP2/TIMP1) was performed. Immunoblotting was performed for expression of α-SMA. In TGF-β1 stimulated HPFB, upregulated expression of pro-fibrotic genes was observed (p<0.05). Expression of pro-fibrotic genes in HPFB cells stimulated with TGF-β1 was significantly (p<0.05) reduced by dual inhibition strategy with complete amelioration of ACTA2 in comparison to their respective individual treatments (figure 1). Ratio of anti-fibrotic genes (MMP2/TIMP1) was restored significantly (p<0.05). In TGF-β1 stimulated HPFB, dual inhibition strategy decreased expression of α-SMA protein significantly (p<0.05). Complete inhibition of α-SMA on dual inhibition strategy of PDE5 and 5-HT2B receptor antagonists suggest inhibition of conversion of resident fibroblasts to MFBs.

SUN-146 5-HT2 AND 5-HT2B RECEPTOR INHIBITION ATTENUATE PERITONEAL FIBROSIS BY TARGETING NON-CANONICAL SIGNALING PATHWAYS INCLUDING STAT3

Peritoneal fibrosis is a major cause of ultrafiltration failure in continuous ambulatory peritoneal dialysis (CAPD) patients. 5-hydroxytryptamine (5-HT; Serotonin) strongly induces extracellular matrix synthesis in peritoneal fibroblasts in a Transforming growth factor beta 1 (TGF-β1) dependent manner. We aimed to evaluate anti-fibrotic role of inhibitors of 5-HT2 and 5-HT2B (Terguride and SB204741), respectively in human peritoneal fibroblasts (HPFB) isolated from peritoneum of CAPD patients.

Evitar (l-Alanyl-l-Glutamine) Regulates Key Signaling Molecules in the Pathogenesis of Postoperative Tissue Fibrosis.

Hypoxia and the resulting oxidative stress play a major role in postoperative tissue fibrosis. The objective of this study was to determine the effect of l-alanyl-l-glutamine (Ala-Gln) on key markers of postoperative tissue fibrosis: hypoxia-inducible factor (HIF) 1α and type I collagen. Primary cultures of human normal peritoneal fibroblasts (NPF) established from normal peritoneal tissue were treated with increasing doses of Ala-Gln (0, 1, 2, or 10 mM) with hypoxia ([2% O2] 0-48 hours; continuous hypoxia) or after hypoxia (0.5, 1, 2, 4 hours) and restoration of normoxia (episodic hypoxia) with immediate treatment with Ala-Gln. Hypoxia-inducible factor 1α and type 1 collagen levels were determined by enzyme-linked immunosorbent assay. Data were analyzed with 1-way analysis of variance followed by Tukey tests with Bonferroni correction. Hypoxia-inducible factor 1α and type I collagen levels increased in untreated controls by 3- to 4-fold in response to continuous and episodic hypoxia in human NPF. Under continuous hypoxia, HIF-1α and type I collagen levels were suppressed by Ala-Gln in a dose-dependent manner. l-alanyl-l-glutamine treatment after episodic hypoxia also suppressed HIF-1α and type I collagen levels for up to 24 hours for all doses and up to 48 hours at the highest dose, regardless of exposure time to hypoxia. l-alanyl-l-glutamine significantly suppressed hypoxia-induced levels of key tissue fibrosis (adhesion) phenotype markers under conditions of continuous as well as episodic hypoxia in vitro. This effect of glutamine on molecular events involved in the cellular response to insult or injury suggests potential therapeutic value for glutamine in the prevention of postoperative tissue fibrosis.

Thy-1+/- fibroblast subsets in the human peritoneum.

Fibrotic thickening of the peritoneum develops in patients receiving peritoneal dialysis (PD) for renal failure. For unknown reasons, however, in some patients it progresses to extensive fibrosis that compromises dialysis capacity of the peritoneum. It is increasingly clear that fibroblasts display large heterogeneity not only between but also within tissues. Differential surface expression of thymocyte differentiation antigen 1 (Thy-1) has been shown to identify functionally distinct fibroblast subsets in several organs. Here, we isolated Thy-1+/- subsets of human peritoneal fibroblasts (HPFB) and analyzed them in terms of profibrotic myofibroblast features. In healthy individuals, Thy-1+ cells constituted ~45% of the HPFB population found in the greater omentum but were not detected in the parietal peritoneum. When propagated in culture and compared with Thy-1- cells, omentum-derived Thy-1+ HPFB consistently displayed an increased expression of α-smooth muscle actin, collagen I, and transforming growth factor-β1. They also showed greater proliferation capacity and enhanced contractile properties. The number of Thy-1+ HPFB increased significantly in PD patients and made up more than 70 and 95% of all HPFB found in the omentum and parietal peritoneum, respectively. These data indicate that the expansion of Thy-1+ fibroblasts may contribute to fibrotic thickening of the peritoneal membrane during PD.

Activation of nuclear factor of activated T cells 5 in the peritoneal membrane of uremic patients.

Peritoneal inflammation and fibrosis are responses to the uremic milieu and exposure to hyperosmolar dialysis fluids in patients on peritoneal dialysis. Cells respond to high osmolarity via the transcription factor nuclear factor of activated T cells (NFAT5). In the present study, the response of human peritoneal fibroblasts to glucose was analyzed in vitro. Expression levels of NFAT5 and chemokine (C-C motif) ligand (CCL2) mRNA were quantified in peritoneal biopsies of five nonuremic control patients, five uremic patients before PD (pPD), and eight patients on PD (oPD) using real-time PCR. Biopsies from 5 control patients, 25 pPD patients, and 25 oPD patients were investigated using immunohistochemistry to detect the expression of NFAT5, CCL2, NF-κB p50, NF-κB p65, and CD68. High glucose concentrations led to an early, dose-dependent induction of NFAT5 mRNA in human peritoneal fibroblasts. CCL2 mRNA expression was upregulated by high concentrations of glucose after 6 h, but, most notably, a concentration-dependent induction of CCL2 was present after 96 h. In human peritoneal biopsies, NFAT5 mRNA levels were increased in uremic patients compared with nonuremic control patients. No significant difference was found between the pPD group and oPD group. CCL2 mRNA expression was higher in the oPD group. Immunohistochemistry analysis was consistent with the results of mRNA analysis. CD68-positive cells were significantly increased in the oPD group. In conclusion, uremia results in NFAT5 induction, which might promote early changes of the peritoneum. Upregulation of NFAT5 in PD patients is associated with NFκB induction, potentially resulting in the recruitment of macrophages.

Regulation of Chemokine CCL5 Synthesis in Human Peritoneal Fibroblasts: A Key Role of IFN-γ

Peritonitis is characterized by a coordinated influx of various leukocyte subpopulations. The pattern of leukocyte recruitment is controlled by chemokines secreted primarily by peritoneal mesothelial cells and macrophages. We have previously demonstrated that some chemokines may be also produced by human peritoneal fibroblasts (HPFB). Aim of our study was to assess the potential of HPFB in culture to release CCL5, a potent chemoattractant for mononuclear leukocytes. Quiescent HPFB released constitutively no or trace amounts of CCL5. Stimulation of HPFB with IL-1β and TNF-α resulted in a time- (up to 96 h) and dose-dependent increase in CCL5 expression and release. IFN-γ alone did not induce CCL5 secretion over a wide range of concentrations (0.01–100 U/mL). However, it synergistically amplified the effects of TNF-α and IL-1β through upregulation of CCL5 mRNA. Moreover, pretreatment of cells with IFN-γ upregulated CD40 receptor, which enabled HPFB to respond to a recombinant ligand of CD40 (CD40L). Exposure of IFN-γ-treated HPFB, but not of control cells, to CD40L resulted in a dose-dependent induction of CCL5. These data demonstrate that HPFB synthesise CCL5 in response to inflammatory mediators present in the inflamed peritoneal cavity. HPFB-derived CCL5 may thus contribute to the intraperitoneal recruitment of mononuclear leukocytes during peritonitis.

Towards Gene Therapy of Postoperative Adhesions: Fiber and Transcriptional Modifications Enhance Adenovirus Targeting towards Human Adhesion Cells

Postoperative abdominal/pelvic peritoneal adhesions are a major source of morbidity (bowel obstruction, infertility, ectopic gestation as well as chronic pelvic pain) in women. In this study, we screened various transduction and transcription modifications of adenovirus (Ad) to identify those that support maximal Ad-mediated gene delivery to human adhesion fibroblasts, which in turn would enhance the efficacy of this novel treatment/preventative strategy for postoperative adhesions. We transduced primary cultures of human peritoneal adhesion fibroblasts with fiber-modified Ad vectors Ad5-RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc as well as transcriptional targeting viruses Ad5-survivin-luc, Ad5-heparanase-luc, Ad5-mesothelin (MSLN)-CRAd-luc and Ad5-secretory leukoprotease inhibitor (SLPI)-luc, and compared their activity to wild-type Ad5-luc. At 48 h, luciferase activity was measured and normalized to the total protein content in the cells. Among the fiber-modified Ad vectors, Ad5-Sigma-luc and among the transcriptional targeting modified Ad vectors, Ad5-MSLN-CRAd-luc showed significantly increased expression levels of luciferase activity at 5, 10 and 50 plaque forming units/cell in adhesion fibroblast cells compared with wild-type Ad5-luc (p < 0.05). Specific modifications of Ad improve their gene delivery efficiency towards human peritoneal adhesion fibroblasts. Developing a safe localized method to prevent/treat postoperative adhesion formation would have a major impact on women health.

Bystander senescence in human peritoneal mesothelium and fibroblasts is related to thrombospondin-1-dependent activation of transforming growth factor-β1

Senescence bystander effect refers to a phenomenon in which senescent cells elicit the development of senescence phenotype in their nearby young counterparts. In this paper we examined the mechanism of senescence bystander effect triggered by senescent human peritoneal mesothelial cells (HPMCs) in proliferating HPMCs and peritoneal fibroblasts (HPFBs). The results showed that conditioned medium (CM) derived from senescent HPMCs elicited a senescence response (growth inhibition coupled with increased expression of senescence-associated β-galactosidase and accumulation of histone γ-H2A.X) in either early-passage HPMCs or HPFBs. Samples of CM from senescent HPMCs contained increased amounts of numerous soluble mediators of which only transforming growth factor-β1 (TGF-β1) was able to induce senescence phenotype in the both types of peritoneal cells, likely through an induction of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (MAPK). At the same time, senescent HPMCs released increased amounts of thrombospondin-1 (TSP-1), a major activator of TGF-β1. Significantly, TSP-1 itself was unable to induce senescence phenotype in HPMCs or in HPFBs. The experiments employing anti-TSP-1 antibodies and specific TSP-1 blocking peptide revealed that neutralization of TSP-1 in CM prevented TGF-β1-dependent development of senescence phenotype. Collectively, our findings indicate that senescent HPMCs exhibit senescence-promoting activity toward neighboring young cells (HPMCs and HPFBs), and this effect is, at least partly, related to TSP-1-dependent activation and further ROS- and p38 MAPK-related activity of TGF-β1.

Effects of hypoxia on the expression of inflammatory markers IL-6 and TNF-a in human normal peritoneal and adhesion fibroblasts

Inflammation is known to be involved in the postoperative adhesion development. Interleukin (IL)-6 and tumor necrosis factor (TNF)-α are cytokines that stimulate the acute-phase reaction, which leads to a systemic reaction including inflammation, fever, and activation of the complement and clotting cascades. The goal of this study was to examine the expression of these inflammatory markers, under normal and hypoxic conditions, in normal and adhesion fibroblasts. Primary cultures of fibroblasts were established from normal peritoneum and adhesion tissues from the same patient(s) and cultured under 20% O2 or hypoxic 2% O2 conditions for 24 hours. Cells were harvested and total RNA was isolated. Complimentary DNA was generated by reverse transcription and subjected to real-time RT-PCR using specific primers for IL-6 and TNF-α. Both normal peritoneal and adhesion fibroblasts expressed IL-6 and TNF-α. Adhesion fibroblasts exhibited significantly higher levels of IL-6 and TNF-α mRNA as compared to normal peritoneal fibroblasts (p < 0.05). Both IL-6 and TNF-α mRNA levels were upregulated in response to hypoxia in both normal peritoneal and adhesion fibroblasts. The increase in IL-6 and TNF-α mRNA levels of normal fibroblasts reached the levels observed in adhesion fibroblasts. Our results suggest that hypoxia promotes the development of the adhesion phenotype by the induction of inflammatory markers, which may contribute to the development of postoperative adhesions. The inhibition of inflammation may be a potential therapeutic approach in the prevention and/or reduction of postoperative adhesion development.

Impact of intraperitoneal pressure of a CO2 pneumoperitoneum on the surgical peritoneal environment†

Animal experiments have suggested that a high intraperitoneal pressure (IPP) might adversely affect the surgical peritoneal environment. The present experimental study investigates the impact of IPP of a CO(2) pneumoperitoneum on human peritoneum. Patients undergoing laparoscopic surgery were subjected to either low (8 mmHg) or standard (12 mmHg) IPP. Normal peritoneum was collected from the parietal wall at the beginning of surgery and every 60 min thereafter. Expression levels of 168 genes that encode extracellular matrix proteins, adhesion molecules or inflammatory cytokine signaling molecules were measured in peritoneal tissues using real-time polymerase chain reaction (PCR)-based assay panels. Human peritoneal mesothelial cells (HPMCs) and human peritoneal fibroblasts (HPFBs) were incubated in a CO(2) insufflation chamber for 1 h at 12 or 8 mmHg. Hyaluronan (HA) synthesis and mRNA expression levels of hyaluronic acid synthases (HAS) and hyaluronidases (Hyal) in HPMCs and HPFBs were measured at 0, 4, 8, 12, 24 and 48 h after CO(2) gas exposure by ELISA and real-time PCR, respectively. Expression levels of connective tissue growth factor (CTGF), matrix metalloproteinase-9, E-selectin, chemokine (C-X-C motif) ligand 2 (CXCL-2), Hyal-1 and Hyal-2 were significantly higher and those of HAS-1, HAS-3, thrombospondin-2 (TSP-2) and interleukin-10 were significantly lower in the 12 mmHg group compared with the 8 mmHg group. HA synthesis was significantly lower in the 12 mmHg group compared with the 8 mmHg group in HPMCs and HPFBs throughout the time course. A low IPP (8 mmHg) may be better than the standard IPP (12 mmHg) to minimize the adverse impact on the surgical peritoneal environment during a CO(2) pneumoperitoneum.

Macrophages induce the adhesion phenotype in normal peritoneal fibroblasts

To determine whether macrophages, exposed to hypoxia, stimulate primary cultures of fibroblasts to acquire the adhesion phenotype. The adhesion phenotype has been previously characterized, in part, by increased fibroblast expression of transforming growth factor (TGF) β1, vascular endothelial growth factor (VEGF), and type I collagen. Media collected from human macrophages cultured under hypoxic conditions (2% O(2)) were used to treat human peritoneal fibroblasts. Additionally, human peritoneal fibroblasts were treated with varying concentrations of TGF-β1. Real-time reverse-transcription polymerase chain reaction and Western blot analysis were used to measure mRNA and protein levels, respectively, for select adhesion markers: TGF-β1, VEGF, and, type I collagen. We hypothesized that macrophage secretion, under hypoxic conditions, is responsible for inducing the adhesion phenotype in human peritoneal fibroblasts. University research laboratory. Human macrophages and peritoneal fibroblasts. Macrophage-fibroblast interaction. Ability of macrophages to induce the adhesion phenotype in human peritoneal fibroblasts. Hypoxia treatment resulted in a significant increase in TGF-β1 expression in human macrophages. Additionally, hypoxia treatment resulted in a significant increase in TGF-β1, VEGF, and type I collagen mRNA and protein levels in normal peritoneal fibroblasts compared with normoxic conditions. Similarly, normal peritoneal fibroblasts treated with media collected from macrophages cultured under hypoxic conditions resulted in a significant increase in TGF-β1, VEGF, and type I collagen mRNA and protein levels compared with normal peritoneal fibroblasts treated with media collected from macrophages cultured under normoxic conditions. Additionally, human peritoneal fibroblasts exposed to varying concentrations of TGF-β1 exhibited a dose-dependent response in the expression of TGF-β1, VEGF, and type I collagen. At a low TGF-β1 concentration (12.5 ng TGF-β1/mL medium), TGF-β1, VEGF, and type I collagen were significantly increased. In contrast, at higher TGF-β1 concentrations (25 and 50 ng TGF-β1/mL media), TGF-β1, VEGF, and type I collagen mRNA levels were significantly reduced compared with 12.5 ng TGF-β1/mL medium. Human macrophages, cultured under hypoxic conditions, release factors that induce the expression of the adhesion phenotype in normal peritoneal fibroblasts. Particularly, TGF-β1 reproduces this response by regulating the expression of TGF-β1, VEGF, and type I collagen in a dose-dependent manner. Therefore, these findings highlight an important role for human macrophages in peritoneal wound healing.

Human adhesion fibroblasts are under constant intrinsic oxidative stress as characterized by higher baseline nadph oxidase and hypoxia inducible factor-1α and lower baseline superoxide dismutase

OBJECTIVE: Recent findings from our laboratory suggest a crucial role for oxidative stress in the pathogenesis of postoperative adhesions. We sought to compare basal levels of oxidative stress markers; NADPH oxidase, hypoxia inducible factor (HIF)-1α, and superoxide dismutase (SOD3), under normal and hypoxic conditions, in human normal peritoneal and adhesion fibroblasts.

Exposure to polychlorinated biphenyls enhances lipid peroxidation in human normal peritoneal and adhesion fibroblasts: A potential role for myeloperoxidase

Nitric oxide, superoxide, and lipid peroxidation (LPO) produced under oxidative stress may contribute to the development of postoperative adhesions. The objective of this study was to determine the effects of polychlorinated biphenyls (PCBs) on LPO, superoxide dismutase, myeloperoxidase (MPO), and nitrite/nitrate in human normal peritoneal and adhesion fibroblasts. PCB treatment reduced inducible nitric oxide synthase (iNOS) expression as well as levels of nitrite/nitrate in both cell lines. Although there was no difference in iNOS expression between the two cell lines, adhesion fibroblasts manifested lower basal levels of MPO compared to normal peritoneal fibroblasts. There was a reduction in MPO expression and its activity in response to PCB treatment in normal peritoneal fibroblasts; however, this effect was minimal in adhesion fibroblasts. Moreover, adhesion fibroblasts manifested higher levels of LPO compared to normal peritoneal fibroblasts, whereas PCB treatment increased LPO levels in both cell types. We conclude that PCBs promote the development of the adhesion phenotype by generating an oxidative stress environment. This is evident by lower iNOS, MPO, and nitrite/nitrate and a simultaneous increase in LPO. Loss of MPO activity, possibly through a mechanism involving MPO heme depletion and free iron release, is yet another source of oxidative stress.