Corneal stroma PDGF blockade and myofibroblast development

Myofibroblast development and haze generation in the corneal stroma is mediated by cytokines, including transforming growth factor-beta (TGF-beta), and possibly other cytokines. This study examined the effects of stromal PDGF-beta blockade on the development of myofibroblasts in response to -9.0 diopter photorefractive keratectomy in the rabbit. Rabbits that had haze generating photorefractive keratectomy (PRK, for 9 diopters of myopia) in one eye were divided into three different groups: stromal application of plasmid pCMV.PDGFRB.23KDEL expressing a subunit of PDGF receptor b (domains 2-3, which bind PDGF-B), stromal application of empty plasmid pCMV, or stromal application of balanced salt solution (BSS). The plasmids (at a concentration 1000 ng/mu l) or BSS was applied to the exposed stroma immediately after surgery and every 24 h for 4-5 days until the epithelium healed. The group treated with pCMV.PDGFRB.23KDEL showed lower alpha SMA+ myofibroblast density in the anterior stroma compared to either control group (P <= 0.001). Although there was also lower corneal haze at the slit lamp at one month after surgery, the difference in haze after PDGF-B blockade was not statistically significant compared to either control group. Stromal PDGF-B blockade during the early postoperative period following PRK decreases stromal alpha SMA+ myofibroblast generation. PDGF is an important modulator of myofibroblast development in the cornea. (C) 2008 Elsevier Ltd. All rights reserved.

Mast cells and transforming growth factor-beta expression: a possible relationship in the development of porphyria cutanea tarda skin lesions

Background Porphyria cutanea tarda (PCT) is a metabolic disease characterized by vesicles and blisters in sun-exposed areas and scleroderma-like lesions in sun-exposed and non-sun-exposed areas. Mast cells participate in the pathogenesis of bullous diseases and diseases that show sclerosis, including PCT. Moreover, transforming growth factor-beta (TGF-beta) is the main cytokine in the development of tissue sclerosis. The correlation of mast cells and TGF-beta with the lesions of PCT has not been examined, however. The possible role of mast cells and TGF-beta (and the relationship between them) in the development of PCT lesions is discussed. Methods To quantify mast cells and cells expressing TGF-beta in skin samples from patients with PCT and controls, immunohistochemical studies were performed in tissue sections allied to morphometric analyses. Results The numbers of mast cells and cells expressing TGF-beta per square millimiter were increased in the PCT group relative to controls, and there was a direct and significant correlation between the mast cell number and cells expressing TGF-beta in PCT. Conclusions The results suggest that the increased number of mast cells and of cells expressing TGF-beta, as well as their direct correlation, may contribute to the pathogenesis of the skin lesions in PCT.

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Treatment With a Growth Factor-Protein Mixture Inhibits Formation of Mineralized Nodules in Osteogenic Cell Cultures Grown on Titanium

Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. This study aimed to evaluate the effects of a well-defined PRP-like mixture containing platelet-derived growth factor-BB, transforming growth factor (TGF)-beta 1, TGF-beta 2, albumin, fibronectin, and thrombospondin [growth factors (GFs) + proteins] on the development of the osteogenic phenotype on titanium (Ti) in vitro. Human alveolar bone-derived osteoblastic cells were subcultured on Ti discs and exposed during the first 7 days to osteogenic medium supplemented with GFs + proteins and to osteogenic medium alone thereafter up to 14 days. Control cultures were exposed to only osteogenic medium. Dose-response experiments were carried out using rat primary calvarial cells exposed to GFs + proteins and 1:10 or 1:100 dilutions of the mixture. Treated human-derived cell cultures exhibited a significantly higher number of cycling cells at days 1 and 4 and of total cells at days 4 and 7, significantly reduced alkaline phosphatase (ALP) activity at days 4, 7, and 10, and no Alizarin red-stained areas (calcium deposits) at day 14, indicating an impairment in osteoblast differentiation. Although the 1:10 and 1:100 dilutions of the mixture restored the proliferative activity of rat-derived osteogenic cells to control levels and promoted a significant increase in ALP activity at day 10 compared with GFs + proteins, mineralized nodule formation was only observed with the 1:100 dilution (similar to 50% of the control). These results showed that a PRP-like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblastic cell cultures grown on Ti. (J Histochem Cytochem 57:265-276, 2009)

Vascular proinflammatory responses by aldosterone are mediated via c-Src trafficking to cholesterol-rich microdomains: role of PDGFR

Aims We demonstrated c-Src activation as a novel non-genomic signalling pathway for aldosterone in vascular smooth muscle cells (VSMCs). Here, we investigated molecular mechanisms and biological responses of this phenomenon, focusing on the role of lipid rafts/caveolae and platelet-derived growth factor receptor (PDGFR) in c-Src-regulated proinflammatory responses by aldosterone. Methods and results Studies were performed in cultured VSMCs from Wistar-Kyoto (WKY) rats and caveolin-1 knockout (Cav 1(-/-)) and wild-type mice. Aldosterone stimulation increased c-Src phosphorylation and trafficking to lipid rafts/caveolae. Cholesterol depletion with methyl-beta-cyclodextrin abrogated aldosterone-induced phosphorylation of c-Src and its target, Pyk2. Aldosterone effects were recovered by cholesterol reload. Aldosterone-induced c-Src and cortactin phosphorylation was reduced in caveolin-1-silenced and Cav 1(-/-) VSMCs. PDGFR is phosphorylated by aldosterone within cholesterol-rich fractions of VSMCs. AG1296, a PDGFR inhibitor, prevented c-Src phosphorylation and translocation to cholesterol-rich fractions. Aldosterone induced an increase in adhesion molecule protein content and promoted monocyte adhesion to VSMCs, responses that were inhibited an by cholesterol depletion, caveolin-1 deficiency, AG1296 and PP2, a c-Src inhibitor. Mineralocorticoid receptor (MR) content in flotillin-2-rich fractions and co-immunoprecipitation with c-Src and PDGFR increased upon aldosterone stimulation, indicating MR-lipid raft/signalling association. Conclusion We demonstrate that aldosterone-mediated c-Src trafficking/activation and proinflammatory signalling involve lipid rafts/caveolae via PDGFR.

Early and late effects of bone marrow-derived mononuclear cell therapy on lung and distal organs in experimental sepsis

We tested the hypothesis that bone marrow-derived mononuclear cells (BMDMCs) at an early phase of cecal ligation and puncture (CLP)-induced sepsis may have lasting effects on: (1) lung mechanics and histology, (2) the structural remodelling of lung parenchyma, (3) lung, kidney, and liver cell apoptosis, and (4) pro- and anti-inflammatory cytokines and growth factors. At day 1, BMDMC significantly reduced mortality, as well as caspase-3, interleukin (IL)-6 and IL-1 beta vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, and transforming growth factor-beta, but increased IL-10 mRNA expression in lung tissue in septic mice contributing to endothelium and epithelium alveolar repair and improvement of lung mechanics. BMDMC also prevented the increase of apoptotic cells in lung, liver, and kidney. At day 7, these early functional and morphological effects were preserved or further improved. In conclusion, in the present model of sepsis, the beneficial effects of early administration of BMDMCs on lung and distal organs were preserved, possibly by paracrine mechanisms. (C) 2011 Elsevier B.V. All rights reserved.

Effects of bone marrow-derived mononuclear cells on airway and lung parenchyma remodeling in a murine model of chronic allergic inflammation

We hypothesized that bone marrow-derived mononuclear cells (BMDMC) would attenuate the remodeling process in a chronic allergic inflammation model. C57BL/6 mice were assigned to two groups. In OVA, mice were sensitized and repeatedly challenged with ovalbumin. Control mice (C) received saline under the same protocol. C and OVA were further randomized to receive BMDMC (2 x 10(6)) or saline intravenously 24 h before the first challenge. BMDMC therapy reduced eosinophil infiltration, smooth muscle-specific actin expression, subepithelial fibrosis, and myocyte hypertrophy and hyperplasia, thus causing a decrease in airway hyperresponsiveness and lung mechanical parameters. BMDMC from green fluorescent protein (GFP)-transgenic mice transplanted into GFP-negative mice yielded lower engraftment in OVA. BMDMC increased insulin-like growth factor expression, but reduced interleukin-5, transforming growth factor-beta, platelet-derived growth factor, and vascular endothelial growth factor mRNA expression. In conclusion, in the present chronic allergic inflammation model, BMDMC therapy was an effective pre-treatment protocol that potentiated airway epithelial cell repair and prevented inflammatory and remodeling processes. (C) 2010 Elsevier B.V. All rights reserved.

Gastrointestinal stromal tumor in Brazil: Clinicopathology, immunohistochemistry, and molecular genetics of 513 cases

The aim of the present study was to evaluate the clinicopathological, immunohistochemical, and molecular genetic features of gastrointestinal stromal tumors in Brazil and compare them with cases from other countries. Five hundred and thirteen cases were retrospectively analyzed. HE-stained sections and clinical information were reviewed and the immunohistochemical expression of CD117, CD34, smooth-muscle actin, S-100 protein, desmin, CD44v3 adhesion molecule, p53 protein, epidermal growth factor receptor, and Ki-67 antigen was studied using tissue microarrays. Mutation analysis of KIT and platelet-derived growth factor receptor-alpha genes was also performed. There was a slight female predominance (50.3%) and the median age at diagnosis was 59 years. The tumors were mainly located in the stomach (38.4%). Immunohistochemistry showed that CD117 was expressed in 95.7% of cases. Epidermal growth factor receptor expression was observed in 84.4% of tumors. p53 protein expression was found only in 2.6% of cases but all belonged to the high-risk group for aggressive behavior according to the National Institutes of Health consensus approach. No CD44v3 adhesion molecule expression was detected. KIT exon 11 mutations were the most frequent (62.2%). The present data confirm that gastrointestinal stromal tumors in Brazilian patients do not differ from tumors occurring in other countries.

Insulin-like growth factor-1 and 17 beta-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells

The PKC apoptosis WTI regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17 beta-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 mu M ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 mu M LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 mu M SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.

Vascular endothelial growth factor and beta-human chorionic gonadotropin are associated with trophoblastic invasion into the tubal wall in ectopic pregnancy

Objective: To assess the association between the depth of trophoblastic penetration into the tubal wall with serum concentrations of vascular endothelial growth factor (VEGF) and beta-hCG and to assess its predictive value. Design: Prospective study. Setting: Tertiary care university hospital. Patient(s): Thirty patients with ampullary pregnancy undergoing salpingectomy were analyzed. Intervention(s): Trophoblastic invasion was histologically classified as stage I when limited to the tubal mucosa, stage II when extending to the muscle layer, and stage III in the case of complete tubal wall infiltration. Main Outcome Measure(s): The relation between depth of trophoblastic infiltration into the tubal wall with VEGF and beta-hCG serum concentrations on the day of surgery. Result(s): An association between the depth of trophoblastic invasion and maternal serum concentrations of VEGF and beta-hCG was observed. VEGF levels of 297.2 pg/mL showed 100.0% sensitivity and 90.0% specificity for stage I, and levels of 440.1 pg/mL showed 81.8% sensitivity and 88.8% specificity for stage III. Beta-hCG levels of 2590.0 mIU/mL showed 88.9% sensitivity and 80.0% specificity for stage I, and levels of 10,827.0 mUI/mL showed 72.7% sensitivity and 88.9% specificity for stage III. Conclusion(s): Maternal serum VEGF and beta-hCG concentrations are associated with depth of trophoblastic penetration into the tubal wall. (Fertil Steril (R) 2010;94:1595-600. (C) 2010 by American Society for Reproductive Medicine.)

In vivo effects of TGF beta 1 on the the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-beta (TGF beta) wanes in the milk of lactating rat, an increase in TGF beta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGF beta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGF beta 1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGF beta 1 while T beta RII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p < 0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p < 0.05). Importantly, TGF beta 1 inhibited cell proliferation (p < 0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p < 0.05). Also, TGF beta 1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p < 0.001), and by morphological features at 12 h (p < 0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGF beta 1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TG beta 1 in gastric growth during postnatal development, (C) 2007 Elsevier B.V. All rights reserved.

Bradykinin B(1) receptor antagonist R954 inhibits eosinophil activation/proliferation/migration and increases TGF-beta and VEGF in a murine model of asthma

In the present study the effects of bradykinin receptor antagonists were investigated in a murine model of asthma using BALB/c mice immunized with ovalbumin/alum and challenged twice with aerosolized ovalbumin. Twenty four hours later eosinophil proliferation in the bone marrow, activation (lipid bodies formation), migration to lung parenchyma and airways and the contents of the pro-angiogenic and pro-fibrotic cytokines TGF-beta and VEGF were determined. The antagonists of the constitutive B(2) (HOE 140) and inducible B(1) (R954) receptors were administered intraperitoneally 30 min before each challenge. In sensitized mice, the antigen challenge induced eosinophil proliferation in the bone marrow, their migration into the lungs and increased the number of lipid bodies in these cells. These events were reduced by treatment of the mice with the B(1) receptor antagonist. The B(2) antagonist increased the number of eosinophils and lipid bodies in the airways without affecting eosinophil counts in the other compartments. After challenge the airway levels of VEGF and TGF-beta significantly increased and the B(1) receptor antagonist caused a further increase. By immunohistochemistry techniques TGF-beta was found to be expressed in the muscular layer of small blood vessels and VEGF in bronchial epithelial cells. The B(1) receptors were expressed in the endothelial cells. These results showed that in a murine model of asthma the B(1) receptor antagonist has an inhibitory effect on eosinophils in selected compartments and increases the production of cytokines involved in tissue repair. It remains to be determined whether this effects of the B(1) antagonist would modify the progression of the allergic inflammation towards resolution or rather towards fibrosis. (C) 2009 Elsevier Ltd. All rights reserved.

Tamoxifen inhibits transforming growth factor-alpha gene expression in human breast carcinoma samples treated with triiodothyronine

Objectives: To examine the effects of triiodothyronine (T(3)), 17 beta-estradiol (E(2)), and tamoxifen (TAM) on transforming growth factor (TGF)-alpha gene expression in primary breast cancer cell cultures and interactions between the different treatments. Methods and results: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3-mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T(3); dish 3: T(3)+TAM; dish 4: TAM; dish 5: E(2); dish 6: E(2)+TAM. TGF-alpha mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T(3) for 48 h significantly increased TGF-alpha mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-alpha mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. Conclusion: We demonstrate that TGF-alpha mRNA expression is more efficiently upregulated by T(3) than E(2). Concomitant treatment with TAM had a mitigating effect on the T(3) effect, while E(2) induced TGF-alpha upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-alpha, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER alpha and beta; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E(2).. Endocrinol. Invest. 31: 1047-1051, 2008) (c) 2008, Editrice Kurtis

High-Resolution Genomic Mapping Reveals Consistent Amplification of the Fibroblast Growth Factor Receptor Substrate 2 Gene in Well-Differentiated and Dedifferentiated Liposarcoma

Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31 CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) ""single nucleotide polymorphism""/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at 1436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma. (C) 2011 Wiley-Liss, Inc.

Topical interleukin-1 receptor antagonist inhibits inflammatory cell infiltration into the cornea

Interleukin (IL)-1 alpha and beta are important modulators of many functions of corneal epithelial and stromal cells that occur following injury to the cornea, including the influx of bone marrow-derived inflammatory cells into the stroma attracted by chemokines released from the stroma and epithelium. In this study, we examined the effect of topical soluble IL-1 receptor antagonist on bone marrow-derived cell influx following corneal epithelial scrape injury in a mouse model. C57BL/6 mice underwent corneal epithelial scrape followed by application of IL-1 receptor antagonist (Amgen, Thousand Oaks, CA) at a concentration of 20 mg/ml or vehicle for 24 h prior to immunocytochemical detection of marker CD11b-positive cells into the stroma. In two experiments, topical IL-1 receptor antagonist had a marked effect in blocking cell influx. For example, in experiment 1, topical IL-1 receptor antagonist markedly reduced detectible CD11b-positive cells into the corneal stroma at 24 It after epithelial injury compared with the vehicle control (3.5 +/- 0.5 (standard error of the mean) cells/400x field and 13.9 +/- 1.2 cells/400x field, respectively, p < 0.01). A second experiment with a different observer performing cell counting had the same result. Thus, the data demonstrate conclusively that topical IL-1 receptor antagonist markedly down-regulates CD-11b-positive monocytic cell appearance in the corneal stroma. Topical IL-1 receptor antagonist could be an effective adjuvant for clinical treatment of corneal conditions in which unwanted inflammation has a role in the pathophysiology of the disorder. (c) 2008 Elsevier Ltd. All rights reserved.