Normal Peritoneal Research Articles

Superoxide induces the adhesion phenotype role of hypoxia in the pathogenesis of the adhesion development

ObjectiveExposure of normal peritoneal fibroblasts to hypoxia has been shown to induce irreversible molecular changes in peritoneal fibroblasts that produce a phenotype, which increases type I collagen and transforming growth factor beta 1 (TGF-b1) expression and thereby would promote adhesion development. This effect is hypothesized to be mediated by superoxide ions generated upon exposure to hypoxia. To determine the effect of xanthene oxidase (XO), a superoxide generating system, and superoxide dismutase (SOD), a superoxide scavenger, on the expression of the adhesion phenotype markers; TGF-b1 and type I collagen.DesignCell Culture Study.Materials and methodsFibroblasts from normal peritoneal and adhesion tissues were cultured with and without SOD (10 U/ml) or XO (14.4 U/ml), which generates superoxide at a rate of 1.4 μM per minute, for 24 hours. Real time RT/PCR was utilized to measure mRNA levels for TGF-b1 and type I collagen.ResultsAs expected, adhesion fibroblasts exhibited significantly higher basal mRNA levels for both TGF-b1 and type I collagen as compared to normal peritoneal fibroblasts. Treatment with XO resulted in a significant increase in both TGF-b1 and type I collagen in normal peritoneal and adhesion fibroblasts. In contrast, treatment with SOD resulted in a significant decrease in both TGF-b1 and type I collagen in normal peritoneal and adhesion fibroblasts.ConclusionsOur results clearly indicate that superoxide is a key player in the formation of the adhesion phenotype. Furthermore, reduction in superoxide levels was shown to significantly reduce the adhesion phenotype markers suggesting a possible mechanism for intervention in the development of the adhesion phenotype. ObjectiveExposure of normal peritoneal fibroblasts to hypoxia has been shown to induce irreversible molecular changes in peritoneal fibroblasts that produce a phenotype, which increases type I collagen and transforming growth factor beta 1 (TGF-b1) expression and thereby would promote adhesion development. This effect is hypothesized to be mediated by superoxide ions generated upon exposure to hypoxia. To determine the effect of xanthene oxidase (XO), a superoxide generating system, and superoxide dismutase (SOD), a superoxide scavenger, on the expression of the adhesion phenotype markers; TGF-b1 and type I collagen. Exposure of normal peritoneal fibroblasts to hypoxia has been shown to induce irreversible molecular changes in peritoneal fibroblasts that produce a phenotype, which increases type I collagen and transforming growth factor beta 1 (TGF-b1) expression and thereby would promote adhesion development. This effect is hypothesized to be mediated by superoxide ions generated upon exposure to hypoxia. To determine the effect of xanthene oxidase (XO), a superoxide generating system, and superoxide dismutase (SOD), a superoxide scavenger, on the expression of the adhesion phenotype markers; TGF-b1 and type I collagen. DesignCell Culture Study. Cell Culture Study. Materials and methodsFibroblasts from normal peritoneal and adhesion tissues were cultured with and without SOD (10 U/ml) or XO (14.4 U/ml), which generates superoxide at a rate of 1.4 μM per minute, for 24 hours. Real time RT/PCR was utilized to measure mRNA levels for TGF-b1 and type I collagen. Fibroblasts from normal peritoneal and adhesion tissues were cultured with and without SOD (10 U/ml) or XO (14.4 U/ml), which generates superoxide at a rate of 1.4 μM per minute, for 24 hours. Real time RT/PCR was utilized to measure mRNA levels for TGF-b1 and type I collagen. ResultsAs expected, adhesion fibroblasts exhibited significantly higher basal mRNA levels for both TGF-b1 and type I collagen as compared to normal peritoneal fibroblasts. Treatment with XO resulted in a significant increase in both TGF-b1 and type I collagen in normal peritoneal and adhesion fibroblasts. In contrast, treatment with SOD resulted in a significant decrease in both TGF-b1 and type I collagen in normal peritoneal and adhesion fibroblasts. As expected, adhesion fibroblasts exhibited significantly higher basal mRNA levels for both TGF-b1 and type I collagen as compared to normal peritoneal fibroblasts. Treatment with XO resulted in a significant increase in both TGF-b1 and type I collagen in normal peritoneal and adhesion fibroblasts. In contrast, treatment with SOD resulted in a significant decrease in both TGF-b1 and type I collagen in normal peritoneal and adhesion fibroblasts. ConclusionsOur results clearly indicate that superoxide is a key player in the formation of the adhesion phenotype. Furthermore, reduction in superoxide levels was shown to significantly reduce the adhesion phenotype markers suggesting a possible mechanism for intervention in the development of the adhesion phenotype. Our results clearly indicate that superoxide is a key player in the formation of the adhesion phenotype. Furthermore, reduction in superoxide levels was shown to significantly reduce the adhesion phenotype markers suggesting a possible mechanism for intervention in the development of the adhesion phenotype.

Nitric oxide synthase isoforms are differentially expressed in fibroblasts isolated from human normal peritoneum and adhesion tissues

There are three isoforms of nitric oxide synthase (NOS), named according to their activity or the tissue type in which they were first described: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). nNOS and eNOS are two constitutive isoforms and iNOS is the induced, more persistent one. The three NOSs catalyze the formation of citrulline and nitric oxide (NO) from L-arginine (L-Arg), NADPH, and O2. Adhesion fibroblasts exhibit a significant reduction in NO levels, although there was no difference in mRNA and protein levels of iNOS between normal peritoneal and adhesion fibroblasts.

Adenovirus-mediated expression of cyclooxygenase-2 antisense reverse abnormal genetic profile of human adhesion fibroblasts

To determine the effects of blocking the translation of cyclooxygenase-2 (COX-2) mRNA on the mRNA levels of type I collagen, type III collagen, fibronectin, and transforming growth factor-beta (TGF-beta1) in fibroblasts obtained from normal peritoneal and adhesion tissues. Prospective experimental study. University medical center. Fibroblasts established from peritoneal and adhesion tissue of the same patients. Adenovirus with COX-2 treatment of the primary cultured fibroblasts. Fibroblasts of normal peritoneal and adhesion tissues were isolated from the same patients. Adhesion and normal peritoneal fibroblasts were transfected with an adenovirus COX-2 mRNA in sense or antisense orientation. RNA was extracted from each treatment and subjected to real time reverse transcriptase-polymerase chain reaction to quantify change in mRNA levels of type I collagen, type III collagen, fibronectin, and TGF-beta1. Type I collagen, type III collagen, fibronectin, and TGF-beta1 mRNAs were present in both normal peritoneal and adhesion fibroblasts with significantly higher levels in adhesion fibroblasts. Normal fibroblasts transfected with the COX-2 sense virus exhibited an elevated mRNA level in those molecules that reached the mRNA levels seen for adhesion fibroblasts. There were no effects seen on the mRNA levels in those molecules when adhesion fibroblasts were transfected with COX-2 sense virus. Normal fibroblasts transfected with COX-2 antisense virus exhibited no difference in mRNA levels in those molecules as compared with untransfected controls. In contrast, adhesion fibroblasts transfected with COX-2 antisense exhibited markedly reduced mRNA levels in those molecules to levels that were seen in normal peritoneal fibroblasts. Our data suggest that inhibition of COX-2 may reduce the development of postoperative adhesions by preventing the formation of the adhesion phenotype.

In this issue

In this issue

Antiadhesion effects of docosahexaenoic acid on normal human peritoneal and adhesion fibroblasts

To determine whether docosahexaenoic acid (DHA) reduces adhesion marker mRNA levels in normal peritoneal and adhesion fibroblasts. Prospective experimental study. University Medical Center. Three patients undergoing laparotomy with excision of adhesions and normal peritoneum. DHA treatment (100 muM) of cell cultures for 24 hours. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) quantification of relative changes (mRNA copies/mug mRNA) in mRNA levels of type I collagen, vascular endothelial growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1). The DHA treatment significantly reduced type I collagen and VEGF, but not TGF-beta1 mRNA levels in normal peritoneal fibroblasts compared to normal controls. The DHA treatment of adhesion fibroblasts reduced type I collagen mRNAs to those of normal peritoneal fibroblasts, decreasing mRNAs by 35% compared to untreated adhesion fibroblasts. The VEGF mRNA levels were 50% lower in DHA-treated adhesion fibroblasts versus untreated adhesion fibroblasts. Docasahexaenoic acid reduced TGF- beta1 mRNA to normal levels in treated adhesion fibroblasts compared to untreated normal peritoneal fibroblasts. Docasahexaenoic acid substantially reduces levels of adhesion-related markers in normal peritoneal and adhesion fibroblasts. This study provides the molecular basis for an easily administered and potentially, highly efficacious, antiadhesion adjuvant.

Modulation of the expression of peroxisome proliferators-activated receptors in human fibroblasts

To determine the levels of peroxisome proliferators-activated receptors (PPARs) in normal and adhesion fibroblasts, we utilized real-time reverse transcription-polymerase chain reaction to measure messenger RNA (mRNA) levels in fibroblasts from normal peritoneum and adhesions from five patients in both the presence or absence of dichloroacetic acid (DCA) and a cyclooxygenase-2 (COX-2) inhibitor, NS-398. Peroxisome proliferators-activated receptor alpha, PPARbeta, PPARgamma1, and PPARgamma2 mRNA are all present in normal peritoneal and adhesion fibroblasts, and selectively rose in response to hypoxia and either DCA or NS-398.

Effects of hyperglycemia on the differential expression of insulin and insulin-like growth factor-I receptors in human normal peritoneal and adhesion fibroblasts

To determine whether the insulin receptor (IR), insulin-like growth factor-I receptor (IGF-IR), and IGF-I are expressed differentially in fibroblasts isolated from normal peritoneal and adhesion tissue before and after 24-hour treatment with increasing glucose concentrations. Prospective experimental study. University medical center. Primary cultures of fibroblasts established from peritoneal and adhesion tissues of the same patients. Glucose treatment of the primary cultured fibroblasts for 24 hours with increasing concentrations of glucose (100-850 mg/dL). Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) levels for IR, IGF-IR, and IGF-I. Enzyme-linked immunosorbent assay was used to determine protein levels. At the normal glycemic level (100 mg/dL), adhesion fibroblasts have significantly higher mRNA levels of the IR (7.96 +/- 0.15 vs. 6.97 +/- 0.16; P<.05), IGF-IR (7.72 +/- 0.22 vs. 6.88 +/- 0.06; P<.05), and IGF-I (7.04 +/- 0.10 vs. 5.92 +/- 0.10; P<.05) when compared with normal fibroblasts, respectively. Data are expressed as log(mRNA/microg RNA). Normal fibroblasts respond to increasing glucose concentrations by increasing the expression levels of the IR, IGF-IR, and IGF-I, whereas adhesion fibroblasts respond by decreasing the expression of the IR, IGF-IR, and IGF-I. The differential expression of the IR, IGF-IR, and IGF-I in adhesion fibroblasts may contribute to the pathogenesis of fibrosis observed in diabetic patients.

O-101: IL6 expression in human normal peritoneal and adhesion fibroblasts: Regulation by hypoxia

We have previously identified phenotypic differences between fibroblasts isolated from normal peritoneum and from adhesions. Specifically, we have shown that adhesion fibroblasts manifest significantly higher levels of the inflammatory cytokines TGF-β1 and type I collagen as compared to normal peritoneal fibroblasts. IL6 is released in response to IL-1 and TNF-alpha and stimulates the acute-phase reaction which leads to a systemic reaction, including inflammation, fever and activation of the complement and clotting cascade. Therefore, we sought to determine the levels of IL6 mRNA in fibroblasts isolated from human normal peritoneum and adhesion tissues under normoxic and hypoxic conditions. Prospective experimental study. Primary cultures of fibroblasts were established from normal peritoneum and adhesion tissues of the same patients (n=3). Fibroblasts were cultured under normal (20% O2) and hypoxic (2% O2) conditions as follow. Hypoxic experiments were performed in an airtight Plexiglas chamber (Bellco Glass, Vineland, NJ). The chamber was deoxygenated by positive infusion of 2% Oxygen in Carbon Dioxide-Nitrogen gas mixture. Cultures were then placed in a standard humidified tissue incubator for 24 hours. Parallel cultures were placed in normoxia for the 24-hour time point. Cells were harvested and total RNA was isolated from each cell type. Complimentary DNA (cDNA) was generated by reverse transcriptase. Aliquots from cDNA were subjected to real-time RT-PCR to measure mRNA levels for IL6, using a QuantitTect SYBR Green kit (Qiagen) and Cepheid 1.2F Detection System. After PCR, a melting curve analysis was performed to demonstrate the specificity of the PCR product as a single peak. The amount of each mRNA was then normalized to the abundance of a housekeeping gene, β-actin. IL6 mRNA was present in both normal peritoneal and adhesion fibroblasts. Adhesion fibroblasts of all patients exhibited significantly higher levels of IL6 mRNA as compared to normal peritoneal fibroblasts (p<0.05). Hypoxia treatment significantly induced IL6 mRNA levels in both normal peritoneal and adhesion fibroblasts. The increase in IL6 mRNA levels of normal fibroblasts reached the levels observed for adhesion fibroblasts (20 fold increase with p<0.05). However, hypoxia had less effect on IL6 mRNA levels in adhesion fibroblasts. The differential expression of IL6 between normal peritoneal and adhesion fibroblasts and the regulation of IL6 by hypoxia suggest a possible inflammatory role in the development of adhesions. Modulation of IL6 may potentially provide the opportunity to reduce postoperative adhesion development.

O-18: TNF-alpha expression in human normal peritoneal and adhesion fibroblasts: Regulation by hypoxia

We have previously identified differences between fibroblasts isolated from normal peritoneum and adhesions, and have identified phenotype differences between these two cell types from the same patients. Specifically, we have shown that adhesion fibroblasts manifest significantly higher levels of the inflammatory cytokines TGF-β1 and type I collagen as compared to normal peritoneal fibroblasts. TNF-alpha is an acute phase protein which initiates a cascade of cytokines and increases vascular permeability, thereby recruiting cells to the site of infection.

Angiogenic factors in peritoneal adhesion formation

Abdominal surgery is considered as the leading cause of peritoneal adhesions and almost universally as adhesiogenic. Peritoneal injury at the time of surgery initiates an inflammatory reaction determining fibrin deposition on the wound surface. Depending on the balance between the different components of the plasminogen system, this fibrin can be either lysed, leading to normal peritoneal healing, or organised, serving as a scaffold for fibroblast ingrowth, extracellular matrix deposition and angiogenesis, leading to adhesion formation. The mechanism underlying the predisposition to form adhesions in some patients and in some specific anatomic sites and not in others after similar surgical procedures remains unknown. In spite of the many attempts proposed over the years for reducing the incidence of adhesion formation, peritoneal adhesions remain a major clinical problem, inducing intestinal obstruction, pelvic pain, female infertility and difficulties at the time of re-operation. The available evidence indicates that understanding the adhesion formation process at the molecular level is essential for developing successful strategies for preventing adhesions. Fortunately, the advancement in molecular biology during the last years has led to the identification of many molecules with the potential of regulating inflammatory and immune responses, tissue remodelling and angiogenesis, key events in peritoneal healing and adhesion formation. This review focuses on the role of angiogenesis and angiogenic factors in peritoneal adhesion formation.

Nonphotochemical Hole-Burning Study of Selectively Stained Normal and Cancerous Human Ovarian Tissues

Results are presented of nonphotochemical hole-burning (HB) experiments on cancerous ovarian and analogous normal peritoneal in vitro tissues stained with the mitochondrial-selective dye rhodamine 800. A comparison of fluorescence excitation spectra, hole-growth kinetics data, and external electric field (Stark) effects on the shape of spectral holes burned in cancerous and normal tissues stained with rhodamine 800 revealed significant differences only in the dipole moment change (fDeltamu) measured by a combination of HB and Stark spectroscopies. It is shown that the permanent dipole moment change for the S0--> S1 transition of the rhodamine 800 molecules in cancerous tissue is higher than that of normal tissue by a factor of about 1.4. The finding is similar to the HB results obtained earlier for human ovarian surface epithelial cell lines, i.e., OV167 carcinoma and OSE(tsT)-14 normal cells stained with the same mitochondria-specific dye (Walsh et al. Biophys. J. 2003, 84, 1299). We propose that the observed difference in the permanent dipole moment change in cancerous ovarian tissue is related to a modification in mitochondrial membrane potential.

Regulation of inducible nitric oxide synthase in post-operative adhesions

The deficiency of the inducible nitric oxide synthase (iNOS) substrate, L-arginine (L-Arg), the co-factor tetrahydrobiopterin (H4B) or molecular oxygen may lead to lower NO levels, which enhances the development of adhesion phenotype. We utilized high-performance liquid chromatography (HPLC) and immunoprecipitation with nitrotyrosine antibody to determine the levels of H4B, citrulline and protein nitration in fibroblasts established from normal peritoneal and adhesion tissues. The level of H4B was dramatically attenuated in adhesion fibroblasts. The immunoprecipitation with nitrotyrosine antibody revealed higher protein nitration in adhesion compared with normal fibroblasts. There were higher accumulations of citrulline in adhesion fibroblasts as compared with normal fibroblasts. In addition, peritoneal fibroblasts treated with 2% oxygen for 24 h and implanted back into the peritoneal cavity of the rats exhibited marked increase in severity of adhesion as well as extensive distribution involving many sites and organs. Control of the catalytic activity of iNOS in adhesion fibroblasts may be because of subsaturating amounts of L-Arg and H4B which allow iNOS to generate a combination of reactive oxygen species in addition to NO, thereby influencing NO bioavailability and function.

196: Cyclooxygenase-2 Inhibitors Enhances Apoptosis Of Adhesion Fibroblasts

Cycloogenase-2 (COX-2), one of the rate limiting enzymes of prostaglandin (PG) synthesis, may play a role in regulating the development of postsurgical adhesions. We have previously reported the COX-2 and PGE2 levels are significantly higher in adhesions as compared to normal fibroblasts. We have also previously shown that normal peritoneal fibroblasts have a higher apoptosis rate as compared to adhesion fibroblasts. We have also found that hypoxia increases COX-2 and PGE2 levels in normal peritoneal fibroblasts and decrease their rate of apoptosis, which may contribute to the formation of postoperative adhesions. The objective of this project is to determine the effects of COX-2 inhibition on the rate of apoptosis of fibroblasts primary cultures established from normal peritoneum and adhesion tissues of the same patient. Fibroblasts from normal peritoneal and adhesion tissues were cultured under normal condition for 24 hours and treated with 100 microM NS398, a COX-2 inhibitor, for 4 hrs before evaluating apoptosis by the TUNEL assay and flow cytometry. University research laboratory. Women with adhesions undergoing gynecologic surgery. NS398 a COX-2 inhibitor. Baseline apoptosis was less in adhesion fibroblasts (2% apoptotic cells) as compared to normal peritoneal fibroblasts (30% apoptotic cells). NS398 treatment resulted in an increase in the rate of apoptosis in adhesion fibroblasts (27% apoptotic cells) but with not effects on normal fibroblasts (31% apoptotic cells). Our data suggests that adhesion fibroblasts may respond to the inhibition of COX-2 by increasing their rate of apoptosis. Inhibition of COX-2 may be important for the elimination of the adhesion fibroblasts during peritoneal healing.

Fluid and Solute Transport in CAPD Patients before and after Permanent Loss of Ultrafiltration Capacity

Two major types of permanent loss of ultrafiltration capacity (UFC) were previously distinguished among patients treated with CAPD: 1) type HDR with high diffusive peritoneal transport rate of small solutes and low osmotic conductance,but with normal fluid absorption rate, and 2) type HAR with high fluid absorption rate, but with normal diffusive peritoneal transport rate of small solutes and normal osmotic conductance. However, the detailed pattern of changes in peritoneal transport parameters in patients developing loss of ultrafiltration capacity is not known. Analysis of solute and fluid transport parameters in the same patient before and after UFC loss. Seven CAPD patients who had undergone repeated dwell studies,which were carried out before and/or after the onset of UFC loss. Dialysis fluids (2 L) with glucose or a mixture of amino acids as osmotic agent at three basic tonicities were applied during 6 hour dwell studies. Fluid and solute transport parameters were previously shown not to be affected by these dialysis solutions (except by hypertonic amino acid-based solution). Intraperitoneal dialysate volume and fluid absorption rate were assessed using radiolabeled human serum albumin (RISA). Osmotic conductance (a(OS))was estimated by a mathematical model as ultrafiltration rate induced by unit osmolality gradient. Diffusive mass transport coefficients, K(BD), for glucose,urea,and creatinine were estimated using the modified Babb-Randerson-Farrell model. Five patients had increased K(BD) for small solutes after the onset of UFC loss,and three of them had decreased a(OS),whereas two patients had normal a(OS). In one of them, a(OS) decreased with time after the onset of UFC loss with concomitant normalization of glucose absorption. In all studies of these five patients the fluid absorption rate was within the normal range. Two other patients had increased fluid absorption rate (about 5 ml/min),and one of them also had increased K(BD) for small solutes,in two consecutive dwell studies in each patient with the second study being carried out at 1 and 7 months respectively after the first one. In all four studies in these two patients, the a(OS) was within the normal range. The sodium dip during dialysis with 3.86% glucose-based solution was lost, not only among most patients with UFC loss related to reduced osmotic conductance, but also in patients with increased K(BD). The occurrence of two major types of UFC loss was confirmed. However, a case of a mixed type of UFC loss with high fluid absorption rate and high K(BD) for small solutes, but normal osmotic conductance, and with normalization of initially high K(BD) for small solutes, linked with decreasing initially normal osmotic conductance,was also found. As a reduced sodium dip with hypertonic glucose solution is not only seen in patients with reduced osmotic conductance, it cannot reliably be used as a single measure of decreased aquaporin function. Permanent ultrafiltration capacity loss may be a dynamic phenomenon with a variety of alterations in peritoneal transport characteristics.

Can Hypoxia Impact Lymphocyte-Mediated Elimination of Peritoneal Fibroblasts Leading to Development of Adhesion Phenotype?

Can Hypoxia Impact Lymphocyte-Mediated Elimination of Peritoneal Fibroblasts Leading to Development of Adhesion Phenotype?

Possible Role of Natural Immune Response Against Fibroblasts in the Development of Post-Operative Adhesions

Intraperitoneal adhesion development following abdominal surgery is a prevalant complication. Fibroblasts obtained from the adhesion tissue (AT) differ from normal peritoneal (NP) fibroblasts in several aspects. Innate immune responses, executed partially by lymphocytes, participate in the elimination of altered cells for the homeostasis of the body. In this study, we investigated NP and AT fibroblast expression patterns of immune response-related markers and lymphokine-activated killer (LAK) cell-mediated fibroblast elimination in vitro.

Knockout of Inducible Nitric Oxide Expression Significantly Reduces the Expression of Type I Collagen and Transforming Growth Factor-Beta 1 in Human Peritoneal and Adhesion Fibroblasts

We have previously identified differences between fibroblasts isolated from normal peritoneum and from adhesions, and have identified phenotype differences between these two cell types from the same patients. Specifically, we have shown that adhesion fibroblasts manifest significantly higher levels of type I collagen and TGF-β1, but with lower nitric oxide levels as compared to normal peritoneal fibroblasts. Our objective is to determine the effects of knocking-out the expression of iNOS in human fibroblasts isolated from normal peritoneal and adhesion tissues on the expression levels of type I collagen and TGF-β1.

Hypoxia upregulates cyclooxygenase-2 and prostaglandin E 2 levels in human peritoneal fibroblasts

To determine the levels of COX-1, COX-2, and prostaglandin (PG) E(2) in human fibroblasts isolated from normal peritoneal and adhesion tissues. Prospective experimental study. University medical center. Fibroblast cultures from both peritoneum and adhesion tissues of five patients. Treatment of the primary cultured fibroblasts with NS398. We used Western blot to determine the effects of hypoxia on COX-1 and COX-2 levels from lysates of normal peritoneal and adhesion fibroblasts before and after hypoxia. We also used the ELISA techniques to determine PGE(2) levels in media collected from these fibroblasts before and after hypoxia and with and without NS398, a COX-2-specific inhibitor. There was no difference in COX-1 levels between normal and adhesion fibroblasts with and without hypoxia. Basal COX-2 and PGE(2) levels were significantly higher in adhesion than normal fibroblasts. Hypoxia gradually increased COX-2 and PGE(2) levels in normal peritoneal fibroblasts over time, reaching a peak at 24 hours but had no effect on adhesion fibroblasts. Inhibition of COX-2 by NS398 significantly reduced PGE(2) levels in both normal and adhesion fibroblasts. The presence of higher levels of COX-2 in adhesion fibroblasts and the induction of COX-2 in normal peritoneal fibroblasts in response to hypoxia indicate a possible inflammatory response. Regulation of COX-2 may alter peritoneal healing and may provide the opportunity to reduce postoperative adhesion development.

Modulation of the expression of vascular endothelial growth factor in human fibroblasts

To examine the up-regulation of vascular endothelial growth factor (VEGF) expression by hypoxia, a crucial event leading to neovascularization, as the reduction in VEGF expression may facilitate minimization of adhesion development. Prospective experimental study. University medical center. Five patients with adhesions undergoing laparotomy with excision of adhesions and normal peritoneum. Adhesion and normal peritoneal fibroblasts were treated with dichloroacetic acid (DCA) or NS-398 (a cyclooxygenase-2 [COX-2] inhibitor) for 24 to 48 hours. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) to quantify relative changes in mRNA levels of VEGF from each treatment. In both normal peritoneal and adhesion fibroblasts, VEGF mRNA was present with statistically significantly higher levels in adhesion fibroblasts (32%). The DCA treatment resulted in a statistically significant decrease in VEGF mRNA levels in adhesion (20%) and normal peritoneal (18%) fibroblasts. The NS-398 treatment resulted in a statistically significant decrease in VEGF mRNA levels in adhesion (25%) and normal peritoneal (16%) fibroblasts. Stimulation of aerobic metabolism by DCA or inhibition of COX-2 by NS-398 reduces VEGF expression. Angiogenesis, which is an integral component in the development of dense vascular adhesions, may be reduced by either COX-2 inhibitors or stimulation of aerobic metabolism by DCA.

Vascular endothelial growth factor a and c gene expression in endometriosis*1

Angiogenesis is essential for the pathogenesis of endometriosis. Gene expression levels of vascular endothelial growth factor (VEGF) A and C in 10 eutopic endometrial, 23 normal peritoneal, and 62 endometriotic tissues surgically obtained from 47 women with endometriosis (group 2) were compared with those in 12 control eutopic endometrial and 9 normal peritoneal tissues from 15 women without endometriosis (group 1). VEGF-A mRNA expression levels in eutopic endometrium of group 2 were higher than those of group 1 throughout the menstrual cycle (P <0.01) and increased in the secretory phase. VEGF-A gene expression in peritoneal endometriotic lesion was statistically higher than that in normal peritoneum (P <0.01) and similar to that in eutopic endometrium of group 2. In contrast, gene expression levels of VEGF-C were relatively lower than those of VEGF-A in each lesion, and no cyclic variation was found. VEGF-A and C mRNA expression levels were significantly higher in ovarian endometriomas >6 cm in size than in those <6 cm in size. Immunohistochemical expression of VEGF-A and C was detected in the cytoplasm of glandular epithelial and stromal cells of ovarian endometrioma. These results suggest that endometriosis may arise from eutopic endometrium with higher levels of angiogenic activity possibly induced by VEGF-A in women with endometriosis. Moreover, VEGF-C as well as VEGF-A may be involved in the pathogenesis of ovarian endometrioma.