Attenuating effects of chymase inhibitor on pericardial adhesion following cardiac surgery

OBJECTIVE: Chymase, a serine protease, is released from mast cells, which is closely associated with adhesion formation. Chymase activates transforming growth factor-beta1 (TGF-beta1), which promotes tissue fibrosis. Recently we have found that chymase may play an important role in adhesion formation in hamsters. Accordingly, this study was designed to confirm that a chymase inhibitor prevents postoperative cardiac adhesions in large animals. METHODS: In 14 dogs, the epicardium was abraded 200 times with gauze and the mid-portion of the left anterior descending coronary artery (LAD) was exposed with No. 15 blade. Either chymase inhibitor (CI group, n = 7) or placebo (P group, n = 7) was sprayed into the pericardial cavity, then the pericardium was closed. Cardiac chymase activity, the level of TGF-beta1 in the pericardial fluid, the density of epicardial mast cells, the adhesion area between the heart and the pericardium, and the presence of adhesion between the mid-LAD and the pericardium were evaluated 1 and 2 months after surgery. Five nonsurgical dogs were used as a control for cardiac chymase activity. RESULTS: Cardiac chymase activity and TGF-beta1 level were lower in CI group than in P group (53.7 +/- 35.0 vs. 93.4 +/- 20.4 microU/mg protein, p = 0.01, 3.2 +/- 0.9 vs. 4.3 +/- 1.1 microg/mL, p = 0.06, respectively). In CI group, the density of mast cells (19 +/- 5 vs. 32 +/- 8 cells/cm, p < 0.01), the adhesion area (2.2 +/- 0.8 vs. 7.5 +/- 1.5 cm2, p < 0.01), and adhesions between the heart and the mid-LAD (0% vs. 57%) were all reduced. CONCLUSION: Chymase inhibitor suppresses cardiac chymase activity and reduces the TGF-beta1 level, resulting in a reduction of cardiac adhesion in a large animal.     

Chymase inhibitor,BCEAB, suppressed peritoneal adhesion formation in hamster

BACKGROUND: Mast cells are closely related to adhesion formation, while it has been unclear which factor in mast cells plays an important role in the development of adhesion formation. To clarify the role of chymase produced from mast cells in adhesion formation, we investigated the preventive effect of a specific chymase inhibitor, BCEAB, on adhesion formation in a hamster experimental model. MATERIALS AND METHODS: Hamsters were administered orally once daily with 100 mg/kg of BCEAB or placebo from the operated day to 1 week after the operation. The uterus was grasped and denuded by a swab. RESULTS: One week after the operation, the scores for adhesion formation in the chymase inhibitor-treated group were significantly decreased in comparison with those in the placebo-treated group (placebo-treated group, 2.80 +/- 0.20; chymase inhibitor-treated group 1.60 +/- 0.31: P < 0.01). The chymase activity in the injured uterus was also significantly suppressed in the chymase inhibitor-treated group (placebo-treated group, 17.3 +/- 2.69 mU/mg protein; chymase inhibitor-treated group 9.60 +/- 0.89: P < 0.05). After scraping the utelus, the level of transforming growth factor-beta in the peritoneal fluid was significantly increased in the placebo-treated group, while it was suppressed to 70% by the treatment with BCEAB. CONCLUSIONS: The specific chymase inhibitor BCEAB may be a useful drug for prevention of adhesion formation.     

Effect of Chymase-Dependent Transforming Growth Factor β on Peritoneal Adhesion Formation in a Rat Model

Purpose To clarify the role of chymase produced from mast cells, which are closely related to adhesion formation, we investigated the preventive effect of a chymase inhibitor on adhesion formation in a rat model.Methods A lesion was created in rats by uterus scraping, and a chymase inhibitor, Suc-Val-Pro-Phe^p(OPh)₂ (10µM), or a placebo was injected into the abdomen. The level of transforming growth factor (TGF-) in the peritoneal fluid was also measured.Results By 2 weeks after the operation, the scores for adhesion formation in the chymase inhibitor-treated group were significantly lower than those in the placebo-treated group, at 1.64 ± 0.34 and 3.27 ± 0.19, respectively (P < 0.01). After scraping the uterus, the level of TGF- in the peritoneal fluid was significantly higher in the placebo-treated group, whereas it was significantly suppressed by the chymase inhibitor.Conclusions Chymase may play an important role in adhesion formation aided by TGF-.     

Association between the expression of mast cell chymase and intraperitoneal adhesion formation in mice

BACKGROUND: Adhesion formation is a major source of postoperative morbidity and mortality. Mast cells and their major protease, chymase, have been shown to participate in the healing process as well as in tissue remodeling. We aimed to identify the role of mast cells in intraperitoneal adhesion formation and to assess whether there is an association between the expression of mast cell chymase and adhesion formation. MATERIALS AND METHODS: Both mast cell-deficient W/W(V) mice and congenic +/+ mice received a standardized lesion produced by cecal scraping and the application of 95% ethanol. Adhesions were assessed blindly 1 week later using a standardized scale. In addition, histamine content, mast cell numbers, and chymase activity in cecum as well as at the healing sites were evaluated before and 7 days after surgical injury. RESULTS: A significant reduction in adhesion formation was seen in mast cell-deficient W/W(V) mice (P < 0.05). In the normal cecum, histamine content did not significantly differ between W/W(V) and +/+ mice. Chymase activity in cecum was detected in control +/+ mice, but not in W/W(V) mice. Mast cell numbers and chymase activity levels at the healing sites of +/+ mice were significantly increased 7 days after surgery. CONCLUSIONS: Our results indicate that mast cells contribute to intraperitoneal adhesion formation in mice, and suggest that chymase originating from mast cells is important in the development of adhesions.     

The effects of ACE inhibition on serum angiotensin II concentration following cardiac transplantation

AIMS: ANGII mediates vascular neointimal formation through smooth muscle cell stimulation and enhanced production of growth factors leading to increased arterial medial layer thickness, which is a characteristic of transplant arteriosclerosis. ACE inhibition is known to be of benefit to patients with cardiovascular risk factors. We aimed to determine the effect of ACE inhibitor therapy on ACE enzymatic activity and serum ANGII levels following cardiac transplantation. METHODS: A total of 43 serum samples from eight transplant recipients were used for analysis. Samples were taken monthly from the date of transplant for the initial 6 months. ANGII was measured using sandwich ELISA. ACE enzymatic activity was measured using spectrophotometric kinetic analysis. RESULTS: There was a significant reduction in ACE enzymatic activity among individuals treated with ACE inhibitor therapy (18.0 +/- 16.6 vs 31.8 +/- 23.4, P = .008). We found significantly higher ANGII serum levels in patients receiving ACE inhibitor therapy compared to those not (2.4 +/- 2.1 vs 8.0 +/- 7.4, P = .002). There was also a significant positive correlation between ACE enzymatic activity and ANGII serum level (coefficient 0.332, P = .03). CONCLUSIONS: Our results suggest an effective ACE independent pathway for ANGII conversion. Chymase can convert ANGI with higher affinity than ACE. Also, chymase is stored in mast cells, which infiltrate the myocardium following transplantation. This data indicate that pharmacological chymase inhibition may be a possible therapeutic strategy following transplantation.     

Effect of transforming growth factor beta on postoperative adhesion formation and intact peritoneum.

Transforming growth factor beta (TGF beta) is an extremely potent chemoattractant for macrophages, mononuclear leukocytes, and fibroblasts. It also acts as a potent stimulant for collagen and fibronectin synthesis and inhibits epithelial cell growth. TGF beta plays an important role in healing many types of wounds, but its role in peritoneal adhesion formation is not known. These studies were performed to determine if TGF beta could affect postoperative wound healing in a rat model. In the first experiment, 20 rats were divided into two groups and received either 2 micrograms TGF beta or control diluent IP daily for 5 days after surgical injury to the uterine horns. The severity of the adhesions were graded 2 weeks postoperatively using a score of 0-3. The TGF beta group showed a higher adhesion score at 2 weeks compared to control, 2.9 +/- 0.34 and 1.6 +/- 0.61, respectively (P less than 0.001). On H&E stained sections of the adhesions, there was an increase in the number of both inflammatory cells and fibroblasts in the TGF beta-treated animals. A comparison trial of bone-derived TGF beta (a gift from Collagen Corporation, Palo Alto, CA) versus recombinant TGF beta (a gift from Oncogen, Seattle, WA) versus control using the same protocol as above showed that both sources of TGF beta were more effective in promoting postoperative adhesions when compared to controls, and there was no difference between TGF beta groups, 3.0 +/- 0 for both TGF beta groups, and 2.2 +/- 0.91 for control (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased plasma chymase concentration and mast cell chymase expression in venous neointimal lesions of patients with CKD and ESRD

The underlying inflammatory component of chronic kidney disease may predispose blood vessels to intimal hyperplasia (IH), which is the primary cause of dialysis access failure. We hypothesize that vascular pathology and markers of IH formation are antecedent to arteriovenous (AV) fistula creation. Blood, cephalic, and basilic vein segments were collected from predialysis chronic kidney disease (CKD) patients with no previous AV access and patients with end-stage renal disease (ESRD). Immunohistochemistry was performed with antibodies against mast cell chymase, transforming growth factor-beta (TGF-β) and interleukin-6 (IL-6), which cause IH. Plasma chymase was measured by ELISA. IH was present in 91% of CKD and 75% of ESRD vein segments. Chymase was abundant in vessels with IH, with the greatest expression in intima and medial layers, and virtually absent in the controls. Chymase colocalized with TGF-β1 and IL-6. Plasma chymase concentration was elevated up to 33-fold in patients with CKD versus controls and was associated with increased chymase in vessels with IH. We show that chymase expression in vessels with IH corresponds with plasma chymase concentrations. As chymase inhibition attenuates IH in animal models, and we find chymase is highly expressed in IH lesions of patients with CKD and ESRD, we speculate that chymase inhibition could have therapeutic value in humans.     

Correlation between mast cell density and myocardial fibrosis in congestive heart failure patients

Besides the well-established role of mast cells in allergic reactions as an important source of vasoactive and proinflammatory products, they have been related to tissue fibrosis and remodelling processes. In a heart failure (HF) animal model, mast cells were shown to synthesize transforming growth factor beta1 and basic fibroblast growth factor in myocardial tissue and were localized to an area with fibrosis. Our objective was to quantify mast cell density in left ventricles from patients with congestive heart failure who were candidates for transplantation and to analyze whether they showed a correlation with the fibrosis level of the same area. METHODS: We obtained myocardial biopsies from 20 patients with end-stage HF secondary to idiopathic dilated cardiomyopathy (iDCM) undergoing heart transplantation and 15 controls (donors without cardiopathy). Mast cells were detected by immunohistochemistry with a human mast cell chymase antibody and fibrosis levels measured with picrosirius red staining of collagen fibrils with later quantification by morphometry. RESULTS: The patients mean age was 51 +/- 3 years. Fibrosis levels in the myocardial sections from patients with congestive HF was three-fold higher than those in control myocardium (12.41 +/- 1.7% vs 3.98 +/- 0.63%, P < .001). Mast cell density correlated with the collagen fraction and could be fitted to a linear regression curve: collagen fraction = 0.78 + 0.05 mast cell density (n = 33, P < .005, R2 = 0.28). CONCLUSION: The elevated collagen fraction present in failing hearts may be the cause of increased stiffness and loss of elasticity that is detected in patients with end-stage HF. Due to the mast cells capacity to synthesize vasoactive and fibrogenic products and the correlation between their density and fibrosis levels, they probably play a role in the ventricular remodelling in HF.     

Mast cell chymase is a possible mediator of neurogenic bladder fibrosis

AIMS: Urinary bladders of patients with myelomeningocele, owing to spina bifida, are often functionally impaired, fibrotic organs. Common to this condition are repeated occurrences of bladder infection and inflammation. Since mast cells have been associated with a fibrogenic response in inflammatory conditions, we investigated the role of mast cell granule product, chymase, as a mediator of myleodysplastic bladder fibrosis. METHODS: Human control and myelodysplastic bladder tissues were stained with Unna's stain and chymase antibody to determine mast cell number and localization. Cell specific localization of collagen mRNAs was determined by in situ hybridization (ISH). In vitro, normal human bladder fibroblasts were treated with recombinant chymase, heparin and inhibitors, and collagen subtype concentration was determined by enzyme linked immunosorbent assay (ELISA). RESULTS: Myelodysplastic bladders were characterized by increased mast cells in the detrusor muscle layer compared to control bladders, as well as mast cell degranulation and increased connective tissue deposition. Both types I and III collagen mRNA localized to fibroblasts surrounding detrusor muscle fascicles, whereas only collagen III mRNA localized to cells within connective tissue infiltrated muscle bundles in myelomeningocele bladder tissue. Chymase treatment of bladder fibroblasts, in vitro, was dose-dependent and resulted in significant increases in both types I and III collagen. Heparin did not alter collagen protein expression, whereas heparin-chymase combination modulated type III collagen expression. Serine protease inhibitor, phenylmethylsulfonlyfluoride, did not inhibit collagen synthesis, whereas denatured chymase resulted in decreased collagenous protein levels. CONCLUSIONS: Bladder fibrosis may be mediated by mast cell chymase stimulation of collagen synthesis.     

The prevention of postoperative intraperitoneal adhesions by tranilast:N-(3′,4′-dimethoxycinnamoyl)anthranilic acid

The development of postoperative intraperitoneal adhesions continues to be a major concern for surgeons. The purpose of this study was to establish a postoperative adhesion model in rats, and to assess the effectiveness of tranilast (N-(3′,4′-dimethoxycinnamoyl)anthranilic acid) in preventing postoperative adhesion formation. The adhesion model was established in 12 male Donryu rats. This involved two essential factors, drying and bleeding. Another 22 male Donryu rats were used to study the prevention of intraperitoneal adhesions. Tranilast was administered orally pre- and postoperatively. Adhesion strength was evaluated by grading, and basic fibroblast growth factor (bFGF) and transforming growth factor-beta-1 (TGF-β1) concentration were measured. Post-operative intraperitoneal adhesions were seen in all rats, but the adhesions in the tranilast group were significantly less severe than those in the control group. Serum bFGF and TGF-β1 levels in the tranilast group were lower at the time of surgery than those in the control group, and bFGF levels were lower at the endpoint of this study in the tranilast group than in the control group. The TGF-β1 levels at the end-point did not differ between the two groups. These findings demonstrated that tranilast significantly reduced postoperative intraperitoneal adhesion formation.     

Effect of chymase activity on skin thickness in the Clawn miniature pig hypertrophic scarring model

Objective: The female red Duroc pig, a heavy and cumbersome animal, is routinely used as an animal model for hypertrophic scarring. Chymase, a chymotrypsin-like serine protease, plays an important role in skin fibrosis. This study aimed to create a lightweight pig hypertrophic scarring model using Clawn miniature pigs, and to investigate the role of chymase in hypertrophic scarring.

Methods: After creating four skin wounds (7.5?×?7.5?cm, depth?=?0.15?cm) in each pig, skin biopsies were performed after 15, 30, 60, 90, 120, and 150?days. Skin thickness, water content, hydroxyproline percentage, chymase activity, and transforming growth factor-beta 1 concentration were measured, and pathological analyses were performed.

Results and conclusions: Both tissue thickness and chymase activity were increased in scar tissue, peaked on day 90 after injury, and then gradually decreased. Peripheral scar tissue showed higher chymase activity than central scar tissue. Neither chymase activity nor transforming growth factor-beta 1 was detected in the surrounding normal skin, whereas central scar tissue showed a high transforming growth factor-beta 1 concentration, peaking on day 15, and decreasing to normal by day 120. We found the Clawn miniature pig to be a useful model for hypertrophic scarring. Chymase activity and skin thickness were well-correlated, suggesting that scars thicken when chymase activity is high.     

Recipient intramuscular gene transfer of active transforming growth factor-beta1 attenuates acute lung rejection

BACKGROUND: Gene transfer into the donor graft has been demonstrated to be feasible in reducing ischemia-reperfusion injury and rejection in lung transplantation. This study was undertaken to determine whether intramuscular gene transfer into the recipient can also reduce subsequent lung graft rejection. METHODS: Brown Norway rats served as donors and F344 rats as recipients. Recipient animals were injected with 10(10) plaque-forming units of adenovirus encoding active transforming growth factor beta1 (group I, n = 6), beta-galactosidase as adenoviral controls (group II, n = 6), or normal saline without adenovirus (group III, n = 6) into both gluteus muscles 2 days before transplantation. Gene expression was confirmed by enzyme-linked immunosorbent assay. Graft function was assessed on postoperative day 5. RESULTS: Successful gene transfection and expression were confirmed by the presence of active transforming growth factor beta1 protein in muscle and plasma. Oxygenation was significantly improved in group I (group I vs II and III, 353.6 +/- 63.0 mm Hg vs 165.7 +/- 39.9 and 119.1 +/- 41.5 mm Hg; p = 0.02 and 0.004). The muscle transfected with the transforming growth factor beta1 showed granulation tissue with fibroblast accumulation. CONCLUSIONS: Intramuscular adenovirus-mediated gene transfer of active transforming growth factor beta1 into the recipients attenuates acute lung rejection as manifested by significantly improved oxygenation in transplanted lung allografts. This intramuscular transfection approach as a cytokine therapy is feasible in transplantation and may be useful in reducing rejection as well as reperfusion injury.     

Mast cells facilitate local VEGF release as an early event in the pathogenesis of postoperative peritoneal adhesions

BACKGROUND: Peritoneal injury sustained at laparotomy may evoke local inflammatory responses that result in adhesion formation. Peritoneal mast cells are likely to initiate this process, whereas vascular permeability/endothelial growth factor (VEGF) may facilitate the degree to which subsequent adhesion formation occurs. METHODS: Mast cell deficient mice (WBB6F1-/-), along with their mast cell sufficient counterparts (WBB6F1+/+), underwent a standardized adhesion-inducing operation (AIS) with subsequent sacrifice and adhesion assessment 14 days later in a blinded fashion. Additional CD-1 and WBB6F1+/+, and WBB6F1-/- mice were killed 2, 6, 12, and 24 hours after operation for measurement of VEGF by ELISA in systemic serum and peritoneal lavage fluid. Two further groups of CD-1 mice underwent AIS and received either a single perioperative dose of anti-VEGF monoclonal antibody (10 mug/mouse) or a similar volume of IgG isotypic antibody and adhesion formation 2 weeks later was evaluated. RESULTS: WBB6F1-/- mice had less adhesions then did their WBB6F1+/+ counterparts (median [interquartile range] adhesion score 3[3-3] vs 1.5[1-2] respectively; P < .003). Local VEGF release peaked 6 hours after AIS in both WBB6F1+/+ and CD-1 mice whereas levels remained at baseline in WBB6F1-/- mice. CD-1 mice treated with a single dose of anti-VEGF therapy during operation had less adhesions than controls (2[1.25-2] vs 3[2.25-3], P = .0002). CONCLUSIONS: Mast cells and VEGF are central to the formation of postoperative intra-abdominal adhesions with mast cells being responsible, either directly or indirectly, for VEGF release into the peritoneal cavity after operation. In tandem with the recent clinical success of anti-VEGF monoclonal antibodies in oncologic practice, our observations suggest an intriguing avenue for research and development of anti-adhesion strategy.     

Prevention of postoperative intrapleural adhesion of the thoracotomy incision by a bioresorbable membrane in the rat adhesion model.

For the purpose of reducing the risk of lung injury while separating adhesions in repeated pulmonary resections, the inhibitory effect of hyaluronate based bioresorbable membrane (HA membrane) on postoperative adhesions was investigated in a new pleural adhesion model in rats. First of all, a novel post-thoracotomy adhesion model in rats was successfully established by using a combination of mechanical, chemical and ischemic injuries of the pleura during operation. After undergoing the same adhesion-inducing procedures, one of two groups was treated with HA membrane inserted between the lung and the parietal pleura, and the other group underwent infusion of saline only. The severity of the adhesion formation was macroscopically lower in the HA membrane-treated rats, and they had favorable mesothelial regeneration microscopically. Additionally, the activity of type 1 plasminogen activator inhibitor (PAI-1) in the intrapleural lavage fluid (ILF) was measured at 24 hours postoperatively because this is the main influence on adhesion formation after surgery. PAI-1 activity was 23.37+/-2.57 U/ml in the saline-treated group and 17.85+/-3.06 U/ml in the HA membrane-treated group. The result suggests that the HA membrane inhibits postoperative adhesion formation through a significant repression of PAI-1 activity.     

Fibrin gel limits intra-abdominal adhesion formation.

Autologous fibrin gel (FG) has recently been reported efficacious in hepatic injury; the effects of fibrin compounds on intra-abdominal adhesion formation is controversial. This study evaluated intra-abdominal adhesion formation in a rabbit devascularization model. Seventeen New Zealand rabbits were anesthetized and laparotomy was done. The uterine horns were abraded to punctate bleeding followed by bilateral uterine devascularization. Treatment consisted of 10 cc saline control (c) or FG applied to the uterine horns. Peritoneal lavage was done at 15 minutes for red blood cell (RBC) analysis. Autopsy was performed at 1 week. Adhesions were graded from grade 0 (no adhesions) to grade III (dense adhesions). Adhesion grading revealed no difference in average adhesion grade between FG and C with small bowel (1.0 +/- 1.3 vs 0.5 +/- 1.0); bladder (2.1 +/- 1.1 vs 2.4 +/- 1.2); or uterus (1.2 +/- vs 2.0 +/- 1.2). Adhesion grade was significantly less in FG compared to C for the colon and the abdominal incision (0.4 +/- 0.5 vs 1.7 +/- 1.1 and 1.2 +/- 1.1 vs 3.0 +/- 1.2; P less than 0.05 by t-test). There were no differences in lavage RBC count between FG and C (13.1 x 106 +/- 4.1 x 10(6) vs 8.7 x 106 +/- 3.2 x 10(6)). Fibrin gel significantly decreased incisional and colonic adhesions and reduced other abdominal adhesion formation by a nonhemostatic dependent mechanism.     

Vascular endothelial growth factor in coronary sinus: evidence for its association with coronary collaterals

OBJECTIVES: To explore whether local growth factors concentration, including vascular endothelial growth factor (VEGF) and transforming growth factor beta one (TGF-beta(1)), influence the formation of coronary collaterals. DESIGN: Thirty-six patients scheduled for coronary angiography received a 6F Goodale-Lubin catheterization to collect blood from the coronary sinus (CS) and right atrium (RA). RESULTS: Patients with coronary collaterals had a higher number of diseased vessels (2.6+/-0.2 vs. 1.4+/-0.3, p = 0.005), higher percentage of severity of stenosis (93+/-2 vs. 48+/-8, P < 0.001) and higher VEGF concentrations in CS (38.9+/-3.9 pg/ml vs. 20.8+/-1.4 pg/ml, P < 0.001) and in RA (31.7+/-3.1 pg/ml vs. 22.0+/-2.3 pg/ml, p = 0.004). There was no significant relationship between coronary collateral formation and TGF-beta(1) concentration. By binary logistic regression analysis, VEGF concentrations in CS (p = 0.030) and stenosis severity (p = 0.042) are correlated positively with collateral formation. CONCLUSIONS: The association between local, endogenous secretion of VEGF and coronary collateral formation is compatible with a paracrine role for this growth factor in pathophysiologic collateral formation.     

Increased levels of surgical adhesions in TGFbeta1 heterozygous mice.

Adhesion formation and fibrosis represent a major complication of surgical intervention. Reducing the morbidity associated with adhesions requires an understanding of the mechanisms underlying their formation. Since increased levels of transforming growth factor-beta1 (TGFbeta1) have been associated with inflammation and adhesion production, we investigated the requirement of TGFbeta1 in peritoneal adhesion formation utilizing mice carrying a targeted disruption of the TGFbeta1 allele. Mice that were either wild-type (+/+), containing two normal alleles of TGFbeta1, or heterozygous (+/-) for the TGFbeta1 null allele received injections of magnesium silicate (talc), and the extent of abdominal adhesions was determined utilizing a standard grading score. Wild-type (+/+) animals had at least twofold more TGFbeta1 protein in peritoneal fluids at 2 h posttrauma compared to heterozygous (+/-) mice (727 vs. 243 pg TGFbeta1/mg protein by enzyme-linked immunosorbent assay (ELISA) in +/+ and +/- mice, respectively), and had significantly less scar and adhesion formation (p < .05) at 7 days posttrauma (1.8 +/- 0.8 vs. 3.4 +/- 1.4, graded from 0 to 5, in +/+ and +/- mice, respectively). These results demonstrate that haploid insufficiency in TGFbeta1 levels can lead to inappropriate matrix and adhesion production during inflammation, and together with previous studies suggest that any perturbation of normal TGFbeta1 levels can modulate the injury response that regulates the extent of adhesion formation.     

Role of mast cells and myofibroblasts in human peritoneal adhesion formation

OBJECTIVE: To study fibroblasts and mast cells in human peritoneal adhesions and to evaluate whether their interaction plays a role in adhesion development. SUMMARY BACKGROUND DATA: Myofibroblasts play a critical role in wound repair/fibrosis. Mast cells influence the formation of peritoneal adhesions in a rat model, and they are modulators of fibroblast functions. METHODS: Peritoneal adhesion biopsies were processed for either histology (H&E, toluidine blue) or immunohistochemistry (tryptase, laminin, collagen type IV and VIII, and alpha-SMA) or grown as explants for obtention of fibroblasts. The effects of mast cell (HMC-1) sonicate and selected mast cell mediators and cytokines on fibroblast proliferation ([ (3)H]thymidine) and collagen synthesis ([ (3)H]proline) and on fibroblast contractile activity (tridimensional collagen lattice) were evaluated. Mast cell mediators influencing fibroblast proliferation were partially characterized by enzymatic susceptibility and FPLC gel filtration column chromatography. RESULTS: Most of the fibroblasts in peritoneal adhesions were identified as alpha-SMA-positive myofibroblasts. Mast cell hyperplasia was observed and more than one third of the mast cells were degranulated. Few mast cells showed a faint staining for laminin or collagen type IV and VIII. Mast cell sonicate increased fibroblast proliferation and contractile activity while decreasing collagen synthesis. Mast cell sonicate proliferating activities were found to be proteinase-sensitive with a molecular weight of more than 158 kd, of approximately 40 kd, and of less than 10 kd. TGF-beta and tryptase enhanced collagen synthesis; TNF-alpha and chymase decreased it. None of the selected mediators increased fibroblast proliferation. CONCLUSIONS: Myofibroblasts are the main connective tissue cells present in human peritoneal adhesions, and mast cells play a direct role in peritoneal adhesion formation.     

Abdominal insufflation decreases blood loss without worsening the inflammatory response: implications for prehospital control of internal bleeding

Abdominal insufflation (AI) by carbon dioxide has been shown to decrease the rate of bleeding in different swine models of abdominal organ injuries. With development of appropriate tools, AI could be used to control bleeding temporarily in the prehospital setting. Concerns have been raised about the inflammatory response to AI, which could damage organs at a later stage despite initial hemostasis. We hypothesized that AI controls bleeding without inducing an unfavorable inflammatory response. An experimental splenic injury was caused in 28 Yorkshire pigs, which were randomized to: 1) standard resuscitation (n = 14) with crystalloids to a mean arterial pressure of 60 mm Hg, or 2) standard resuscitation and AI (n = 14) to an abdominal pressure of 20 cmH2O. The experiment lasted for 30 minutes, and intra-abdominal blood loss was measured. Blood serum interleukin 1beta (IL-1beta), transforming growth factor beta1, and lung tissue heat shock protein 70 gene expression were measured at 0, 15, and 30 minutes, as markers of the inflammatory response. All animals survived to the end of the experiment. Total blood loss was significantly less in the AI group compared with the other standard resuscitation animals (733 +/- 76 vs 1094 +/- 153 mL, P = 0.049). The pH at the end of the experiment was significantly lower in the AI group (7.28 +/- 0.02 vs 7.44 +/- 0.05, P < 0.01) but there was no difference in lactate levels (1.5 +/- 0.4 vs 1.7 +/- 0.3, P = 0.7). Similarly, there was no difference in IL-1beta, transforming growth factor beta1, or lung tissue heat shock protein 70 gene expression between the two groups at any time point, although there was a trend towards lower IL-1beta levels in the AI group. Our conclusion is that AI reduces blood loss from splenic injury without a measurable effect on the early inflammatory response in a clinically relevant animal model.     

Cytomegalovirus enhance expression of growth factors during the development of chronic allograft nephropathy in rats

Cytomegalovirus (CMV) accelerates chronic rejection (CRX) in a model of rat kidney allograft. In this model, the expressions of transforming growth factor beta 1 (TGF-beta), platelet-derived growth factor (PDGF)-AA, PDGF-BB and connective tissue growth factor (CTGF) were investigated with and without CMV. Transplantations were performed under immunosuppression. One group of animals was infected with CMV and the other was left uninfected. The grafts were harvested on days 3-60 after transplantation. Growth factor proteins were demonstrated by immunohistochemistry, and mRNAs by in situ hybridization. A significantly more intense and earlier endothelial TGF-beta (2.4 +/- 0.8 vs. 1.0 +/- 0.0; P < 0.05) and PDGF-AA (1.8 +/- 0.4 vs. 1.0 +/- 0.0; P < 0.05) expressions, confirmed by mRNA hybridization, occurred in the CMV group compared with the noninfected group. PDGF-BB appeared in a few inflammatory cells only. In addition CTGF appeared earlier and has more intense in the CMV group (2.5 +/- 0.6 vs. 1.2 +/- 0.5) and the number of CTGF mRNA-positive fibroblasts (57 +/- 9 vs. 3 +/- 4; P < 0.05) was significantly higher. Thus, CMV enhanced expression of TGF-beta1, PDGF-AA and CTGF during the development of CRX.