Effect of epidermal growth factor on pulmonary hypoplasia in experimental diaphragmatic hernia

Background/purpose

Currently, tracheal occlusion (TO) is a potent stimulus for fetal lung growth but also a rather invasive and high-risk procedure. The aim of this study was to investigate a new and much less invasive therapeutic strategy, namely the maternal intraperitoneal administration of epidermal growth factor (EGF) and its effect on pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) rat model, especially its effect on type II pneumocytes.

Methods

CDH was induced by maternal administration of a single oral dose (100 mg) of nitrofen on day 8.5 of pregnancy. Four groups of pregnant rats were designed on day 18.5: normal control (n = 4), CDH (n = 4), CDH plus Dex (n = 4), CDH plus EGF (n = 8). All fetuses were delivered by cesarean section on day 21. Accordingly, there were 4 groups of fetuses: normal controls (n = 33), nitrofen-induced CDH (n = 19), CDH plus Dex treatment (n = 15), and CDH plus EGF treatment (n = 24). Lung tissue weight (LW) and body weight (BW) of each fetus were recorded, lung histologic and morphometric evaluations were performed, and image analysis was combined after lung processing. Transmission electron microscopy was used for ultrastructural observation, especially type II pneumocytes.

Results

CDH was observed in 58 of the 94 rat fetuses (61.7%). Lw/Bw of CDH group was significantly lower than those of Dex and EGF (P < .05). The lungs of CDH fetuses showed marked hypoplasia, in contrast to improved mesenchymal differentiation in that of Dex and EGF fetuses. Statistical differences of these morphologic parameters (RAC, MTBD, interstitial%, and alveoli%) were found (P < .05). As to ultrastructural features, type II cells of CDH lungs had few if any lamellar bodies and cytoplasmic organelles, and showed evidence of abundant glycogen granules. The sparse type II cells also showed cytoplasmic degenerative changes. By contrast, type II cells of EGF lungs showed numerous mitochondria, abundant lamellar bodies (surfactant) and deficiency of glycogen granules, and displayed prominent microvillous projections and pitlike depressions. The density of type II pneumocyte were 65 ± 4.5, 31 ± 3.1, and 8 ± 1.5 for EGF, Dex, and CDH, respectively (EGF v Dex, P < .05; EGF v CDH, P < 0.01).

Conclusions

Compared with TO, prenatal EGF administration as a much less-invasive therapeutic strategy had shown marked improvement in pulmonary hypoplasia and promotion of type II pneumocyte differentiation in the nitrofen-induced CDH rat model. Thus, EGF could improve the prognosis of CDH by means of promoting pulmonary hypoplasia and improving the surfactant deficiency, which suggested a potential role in the clinical treatment of CDH.     

Modulation of the inflammatory response and apoptosis using epidermal growth factor and hepatocyte growth factor in a liver injury model: a potential approach to the management and treatment of cholestatic liver disease

Background/Purpose

The major side effect of total parenteral nutrition is liver injury leading to liver failure. This study was designed to assess specific growth factors in modulating the hepatic response in an ANIT-induced liver-injury model.

Methods

Sprague-Dawley rats were divided into four groups: control (n = 5), liver-injury control (α-naphtylisocyocyanate [ANIT], 100 mg/kg, n = 8), ANIT + epidermal growth factor (EGF, 150 μg/kg per day, n = 10), and ANIT + hepatocyte growth factor (HGF, 250 μg/kg per day, n = 9). Rats were given intraperitoneal injections of saline (control) or ANIT and implantation of an osmotic mini-pump for 7 days of continuous intravenous saline (liver injury control), EGF, or HGF. Seven and 14 days later, liver biopsies were obtained and evaluated for interleukin (IL)-6 and tumor necrosis factor α expression by immunofluorescent staining, and for apoptosis, by the terminal transferase dUTP nick end labeling (TUNEL) technique. All animals were euthanized at 14 days.

Results

Epidermal growth factor (P < .025) and HGF (P < .001) groups induced less IL-6 expression at day 14 compared to liver-injury controls. In addition, the interval decrease in IL-6 expression between days 7 and 14 was greater in EGF (P < .001) and HGF (P < .001) groups compared to liver-injury controls. At day 14, HGF also demonstrated decreased tumor necrosis factor α expression (P < .005). Apoptotic activity was significantly less for the EGF (P < .011) and HGF (P < .0012) groups.

Conclusion

Epidermal growth factor and HGF modulated the hepatic inflammatory response and apoptotic index in this established liver-injury model and may diminish or prevent liver damage in patients with total parenteral nutrition-induced liver injury.     

Effect of antenatal tetrandrine administration on endothelin-1 and epidermal growth factor levels in the lungs of rats with experimental diaphragmatic hernia

Purpose

The aim of this study was to evaluate the effect of the traditional Chinese medicine tetrandrine (Tet) and to determine its possible mechanism on expression of endothelin-1 (ET-1) and epidermal growth factor (EGF) in the lung of a rat model of nitrofen-induced congenital diaphragmatic hernia (CDH).

Methods

A single oral dose (115 mg/kg) of nitrofen on day 9.5 of pregnancy was maternally administered to induce CDH. Pregnant rats were divided into 4 groups on day 18.5: control (n = 5), CDH (n = 5), CDH+dexamethasone (Dex) (n = 5), and CDH+Tet (n = 5). All fetuses were delivered by cesarean delivery on day 21.5. Accordingly, there were 4 groups of fetuses: control (n = 38), CDH (n = 25), CDH+Dex (n = 21), and CDH+Tet (n = 22). Lung tissue weight (LW) and body weight (BW) of each fetus were recorded, lung histologic evaluations and ET-1 and EGF immunohistochemistry staining were performed, and image analysis was performed after lung processing.

Results

Five female rats in the control group produced 38 fetuses without CDH. CDH was observed in 68 of the 128 rat fetuses (53.1%) among the other 3 groups. The LW/BW ratio of the CDH group was significantly lower than those of the Dex and EGF groups (P < .05). The lungs of fetuses with CDH showed marked abnormal structure such as pulmonary hypoplasia and vascular remodeling, in contrast to improved pulmonary structure in lungs of fetuses in the CDH+Dex and CDH+Tet groups. Statistical differences in morphologic parameters (radial alveolar counts, percentage of alveoli, percentage of medial wall thickness, and vascular volume) were found (P < .05). The immunoreactivity of EGF and ET-1 in the CDH group was markedly stronger than that in the control, CDH+Dex, and CDH+Tet groups (P < .01). In addition, EGF and ET-1 expression in the CDH+Dex and CDH+Tet groups was stronger than that in the control group (P < .05). There was no difference in lung EGF and ET-1 immunoreactivity between CDH+Dex and CDH+Tet groups (P > .05).

Conclusion

Antenatal treatment with Tet may improve lung growth and vascular remodeling, and its mechanism seems to be involved in decreasing EGF and ET-1 expression. Tet administered maternally may be a hopeful new therapeutic option in the treatment of CDH and may be effective in helping to avoid the side effects of Dex.     

Decreased Expression of Thrombospondin-1 in Failing Hearts May Favor Ventricular Remodeling

Background

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis and an activator of tissue transforming growth factor-β1 (TGF-β1). Analyses using genetically modified mice suggested that TSP-1 may play a protective role to prevent infiltration and tissue remodeling responses after myocardial infarction. The expression levels of TSP-1 and their putative role in ventricular remodeling have not been determined in patients with heart failure (HF).

Materials and Methods

We analyzed the expression of TSP-1 and TGF-β1 mRNA in myocardial biopsies from 34 subjects with end-stage HF undergoing heart transplantation and 13 healthy controls from heart donors. Among total RNA extracted from the left ventricle, 1 μg was retrotranscribed and mRNA expression levels were quantified by real-time polymerase chain reaction (PCR).

Results

The mean age of subjects was 54 ± 2 years; mean ejection fraction, 21 ± 5%; end-diastolic diameter and end-systolic diameter, 73 ± 10 and 61 ± 11 mm, respectively. TSP-1 mRNA expression in ventricular tissue from HF patients was lower (159.04 ± 14.55 ng-equivalents [ng-equiv]) than in controls (234 ± 30.66 ng-equiv; P < .05). Tissue from HF subjects also showed lower levels of TGF-β1 (68.42 ± 4.36 vs 80.58 ± 5.26 ng-equiv; P < .05). TSP-1 mRNA levels correlated positively with TGF-β1 (P = .001; R² = .2), and lower TSP-1 mRNA levels were observed with increasing left ventricular diameters.

Conclusions

Patients with end-stage HF show decreased TSP-1 mRNA levels, which agrees with published results showing lower circulating TSP-1. Ventricular dilatation observed in these patients may be related to lower expression of TSP-1. Surprisingly, TGF-β1 mRNA levels were lower in failing hearts, which suggested that fibrogenesis takes place in earlier phases of HF.     

Effect of phototherapy on growth factor levels in neonatal rat skin

Aim

Neonates undergoing surgery may receive phototherapy (PT) for the treatment of hyperbilirubinemia. Although the effects of PT on neonatal structures are well documented, the effect of PT on wound healing has not been previously evaluated. An experimental study was performed to evaluate the effect of PT on growth factor levels responsible for wound healing in neonatal rat skin.

Materials and Methods

Eighteen Wistar newborn rats (7 ± 2 g) were included in the study. Rats were randomized into 3 groups: control (CG), PT, and sham (SG) (n = 6). Both groups had 1-cm median dorsal skin incision. In CG, 1 × 1 cm of dorsal skin was sampled including the incised skin. The PT group received 5 banks of blue light (wave density, 30-40 μw/cm² per nanometer; exposure distance, 45 cm). Phototherapy was started 24 hours after birth and exposed during light period (mean duration, 21 hours to 15 minutes ± 2 hour to 1.5 minutes). Sham group consisted of animals that received a bank of white light with same exposure distance and a total duration of 26 hours to 18 minutes ± 3 hours to 9.1 minutes. After exposure, 1 × 1 cm dorsal skin samples were obtained from both PT and SG groups, including the median incision. The effect of PT was evaluated with the expressions of vascular endothelial growth factor (VEGF), its receptor (VEGF receptor), and transforming growth factor β (TGF-β) in endothelial vessels and fibroblasts of neonatal skin samples.

Results

There was no significant difference between groups in VEGF receptor and transforming growth factor β expressions. The VEGF levels in endothelial vessels were significantly decreased in PT and SG when compared with CG (P < .05).

Conclusion

Vascular endothelial growth factor is a mediator of angiogenesis and may decrease in neonatal rat skin after light exposure. It can be suggested that decreased levels of VEGF after PT application may alter angiogenesis and also may adversely affect the healing features of neonatal skin.     

Renal protective effects of leukotriene receptor blockers in an experimental model of cyclosporine nephrotoxicity

Background

Chronic cyclosporine (CsA) nephrotoxicity is associated with renal fibrosis and hyaline arteriolopathy. Fibrogenic cytokines, such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF), play a pivotal role in CsA nephrotoxicity. Previous studies have demonstrated the possible role of leukotrienes (LT) in chronic CsA nephrotoxicity. The aim of this study was to examine the possible beneficial effects of LT blockers in attenuating the morphological and histochemical effects induced by CsA in a rat model of CsA nephrotoxicity.

Materials and Methods

Twenty-four male Wistar rats were divided into 3 groups (n = 8). The first group (G1) was treated with vehicle intraperitoneally (IP) for 60 days. The second group (G2) was treated with 15 mg/kg CsA IP for 60 days. The third group (G3) was treated with the same dose of CsA plus 4 mg/kg montelukast administered by oral gavage for 60 days.

Results

There was a statistically significant decrease in glomerular filtration rate (GFR) among G2 compared with G1 animals: 0.41 ± 0.03 vs 1.63 ± 0.12 mL/min (P < .001), or G3 hosts: 0.41 ± 0.03 vs 0.95 ± 0.05 mL/min (P < .005), respectively. The percentage of hyaline arteriolopathic changes was higher in G2 than G1 or G3: 81.66% ± 8.2% vs 11.83% ± 0.87% (P < .01) or 37.0% ± 8.8% (P < .01), respectively. Fibrosis score was higher in G2 compared with G1 or G3: 1.5 ± 0.04 vs 0.16 ± 0.02 (P < .001) and 1.0 ± 0.05 (P < .05), respectively. TGF-β and VEGF immunoexpression were significantly increased in G2 compared with G1 (P < .05) or G3 (P < .05).

Conclusions

Our study suggested that LT may play a critical role in the pathogenesis of chronic CsA nephrotoxicity; the administration of montelukast, a LT receptor blocker, may prevent CsA-induced nephrotoxicity.     

Basic fibroblast growth factor and granulocyte colony-stimulating factor enhance mucosal surface expansion after adult small bowel transplantation without vascular reconstruction in rats

Aim

We showed previously that adult small bowel could be transplanted successfully in rats without vascular reconstruction by removing the graft serosa. In this study, we assessed if granulocyte colony-stimulating factor (G-CSF) or basic fibroblast growth factor (bFGF) could improve graft survival in the same rat model.

Method

A 10-mm-long adult small bowel graft from an adult 12-week-old Lewis rat was transplanted into a pouch created in the omentum of a 5-week-old Lewis rat (syngeneic bowel transplantation [SBTx], n = 49). Graft serosa was removed just before SBTx in the serosectomy group (n = 29) and left intact in the nonserosectomy group (n = 20). Each group was divided into 3 subgroups (sG): sG-1 had no G-CSF or bFGF; sG-2 had daily subcutaneous injections of G-CSF; and sG-3 had continuous infusion of bFGF around the graft in the omentum. All grafts were harvested 14 days after SBTx and studied histologically. A mucosal surface expansion score (MSES) was used where 0 = no mucosa on the graft, 1 = mucosa on one fourth of the graft, 2 = mucosa on one half of the graft, 3 = mucosa on three fourths of the graft, and 4 = mucosa on the whole graft. The density of CD34-positive capillaries per 1000 nuclei was also measured.

Results

Serosectomy group MSES were significantly higher than nonserosectomy group MSES indicating that grafts survived (P < .0001). CD34-positive capillaries in serosectomy group subgroups for mucosa were 103.9 ± 34.2, 130.2 ± 52.0, and 132.3 ± 37.7, respectively; for muscle, 74.4 ± 38.0, 86.2 ± 32.9, and 82.4 ± 30.3, respectively; and for omentum, 73.8 ± 30.1, 151.3 ± 60.3, and 140.0 ± 49.0, respectively. Mucosal surface expansion score and overall CD34-positive capillaries for sG-2 and sG-3 were significantly higher than for sG-1 (both, P < .05).

Conclusion

Our results suggest that G-CSF and bFGF enhance angiogenesis and mucosal surface expansion.     

The effects of local and sustained release of fibroblast growth factor on wound healing in esophageal anastomoses

Background/Purpose

Postsurgical complications, such as anastomotic leaks in patients with esophageal atresia, have remained unchanged during the last 3 decades. Growth factors enhance healing in several wound-healing models. Therefore, an experimental study was used to evaluate the effects of local and sustained release of basic fibroblast growth factor (FGF) on wound healing in esophageal anastomoses.

Materials and Methods

Twenty-four male Wistar albino rats, which were subjected to a 1-cm segmental resection of the abdominal esophagus followed by end-to-end anastomosis, were allocated into 3 groups. Group I, the control group, had no gelatin film applied to the anastomosis. In group II (gelatin film without FGF) and group III (gelatin film with FGF), anastomoses were covered with unloaded and 2.55 μg FGF-loaded gelatin films, respectively. On postoperative day 7, bursting pressures, histopathologic collagen deposition, and tissue hydroxyproline concentrations of the anastomoses were then analyzed and compared.

Results

Mean bursting pressures, mean submucosal and muscular collagen deposition scores, and mean tissue hydroxyproline concentrations differed significantly between groups. Mean bursting pressures were 22.5 ± 3.1 mm Hg in group I, 29 ± 1.6 mm Hg in group II, and 63.2 ± 6.8 mm Hg in group III (P < .001). Mean submucosal collagen deposition scores (group I: 0.7 ± 0.2, group II: 0.7 ± 0.1, group III: 1.5 ± 0.2; P = .02) and mean muscular collagen deposition scores (group I: 0.8 ± 0.2, group II: 0.8 ± 0.1, group III: 1.8 ± 0.1; P = .01) were significantly higher in FGF animals than the other in the other 2 groups. Mean tissue hydroxyproline concentrations were 2.4 ± 0.5 μg/mg in group I, 3.9 ± 0.4 μg/mg in group II, and 6.0 ± 1.0 μg/mg in group III (P = .007).

Conclusion

Local and sustained release of FGF enhanced wound healing in esophageal anastomoses in this animal model.     

Protective effect of estrogen on intestinal ischemia-reperfusion injury in pubertal rats

Purpose

This study investigated the role of 17β-estradiol (E₂) in intestinal ischemia-reperfusion (I/R) injury and its possible mechanism.

Methods

Rats of pubertal age were ovariectomized and injected subcutaneously with vehicle (vehicle group) or E₂ (100 or 500 μg/kg/every other day, E₂ or 5E₂ group) for 4 weeks. Other rats of the same age underwent sham ovariectomy as a control group. The rats in each group (n = 15) were subjected to superior mesenteric artery occlusion followed by 1 hour (n = 5), 6 hours (n = 5), or 24 hours (n = 5) reperfusion. Intestine specimens then were obtained for the determination of histopathologic score, inducible nitric oxide synthase (iNOS) mRNA expression, and iNOS activity.

Results

In vehicle, control, E₂, and 5E₂ groups, the histopathologic scores were 3.31 ± 0.12, 3.00 ± 0.09, 2.57 ± 0.12, and 2.98 ± 0.08, respectively. The expression levels of iNOS mRNA were 3.85 ± 0.42, 4.86 ± 0.76, 5.17 ± 0.34, and 4.25 ± 0.41 log copies, respectively. Lower histopathologic score but higher iNOS mRNA expression were found in E₂ group than in the other groups (P < .01). The level of iNOS activity paralleled the expression of iNOS mRNA.

Conclusions

Estrogen may exert a protective effect on intestinal I/R injury in pubertal rats, probably by enhancing iNOS expression.     

The enhancing role of vitamin A deficiency on chemically induced nephroblastoma in rats

Purpose

Vitamin A plays an important role in the prevention of neoplasia. The occurrence of nephroblastoma, a common renal malignancy of childhood, is said to be closely correlated with retardation of differentiation of metanephric blastema. The aim of this study was to clarify the effect of vitamin A deficiency (VAD) in experimental nephroblastoma.

Methods

Sixty weaning outbred female Wistar rats weighing 40 to 50 g were injected with 0.35% dimethylhydrazine dihydrochloride (DMH) subcutaneously and divided into 2 groups. Animals were fed with diets containing vitamin A, normal diet (ND group; n = 30), and vitamin A-deficient diet (VAD group; n = 30). Serum concentrations of vitamin A on day 120 after DMH injection were assayed. The incidence of nephroblastomas in 2 groups was recorded. The renal tissues were prepared for morphological studies and WT1 messenger RNA (mRNA) detection on day 365 after DMH injection.

Results

The average serum concentrations of vitamin A in the ND group (2.60 ± 0.53 μmol/L) were significantly higher than that in the VAD group (0.51 ± 0.26 μmol/L, P < .05) 120 days after DMH injection. On day 365, the incidence of renal tumors in the VAD group (36.7%) was significantly higher than that of the ND group (13.3%, P < .05). Certain histological and ultrastructural features and WT1 mRNA expression patterns observed in these tumors showed that they had a resemblance to human nephroblastomas. Nephrogenic rests (NRs) could be seen in the adjacent normal renal parenchyma. The incidence of NRs in the VAD group (16.7%) was significantly higher than that in the ND group (3.3%, P < .05). The mutant WT1 mRNA expression levels in the VAD group (0.75 ± 0.11) were significantly higher than those in the ND group (0.24 ± 0.06, P < .05).

Conclusions

Vitamin A deficiency can increase development of nephroblastomas in rats, probably via a mechanism of elevated NR remnants and WT1 expression.