Investigation of the Antihypertrophic and Antifibrotic Effects of Losartan in a Rat Model of Radiation-Induced Heart Disease.

Radiation-induced heart disease (RIHD) is a potential late side-effect of thoracic radiotherapy resulting in left ventricular hypertrophy (LVH) and fibrosis due to a complex pathomechanism leading to heart failure. Angiotensin-II receptor blockers (ARBs), including losartan, are frequently used to control heart failure of various etiologies. Preclinical evidence is lacking on the anti-remodeling effects of ARBs in RIHD, while the results of clinical studies are controversial. We aimed at investigating the effects of losartan in a rat model of RIHD. Male Sprague-Dawley rats were studied in three groups: (1) control, (2) radiotherapy (RT) only, (3) RT treated with losartan (per os 10 mg/kg/day), and were followed for 1, 3, or 15 weeks. At 15 weeks post-irradiation, losartan alleviated the echocardiographic and histological signs of LVH and fibrosis and reduced the overexpression of chymase, connective tissue growth factor, and transforming growth factor-beta in the myocardium measured by qPCR; likewise, the level of the SMAD2/3 protein determined by Western blot decreased. In both RT groups, the pro-survival phospho-AKT/AKT and the phospho-ERK1,2/ERK1,2 ratios were increased at week 15. The antiremodeling effects of losartan seem to be associated with the repression of chymase and several elements of the TGF-ß/SMAD signaling pathway in our RIHD model.

Myostatin Upregulation in Patients in the Chronic Phase of Severe Burn Injury Leads to Muscle Cell Catabolism.

BACKGROUND: Burn injury leads to a hypercatabolic response and ultimately muscle wasting with drastic implications for recovery of bodily functions, patient's quality of life (QoL), and long-term survival. Several treatment options target the body's initial stress response, but pharmacological approaches to specifically address muscle protein metabolism have only been poorly investigated. OBJECTIVE: The aim of this study was to assess the role of myostatin and follistatin in burn injury and its possible implications in muscle wasting syndrome. METHODS: We harvested serum from male patients within 48 h and again 9-12 months after severe burn injury (>20% of total body surface area). By means of myoblast cultures, immunohistochemistry, immunoblotting, and scratch assay, the role of myostatin and its implications in post-burn muscle metabolism and myoblast proliferation and differentiation was analyzed. RESULTS: We were able to show increased proliferative and myogenic capacity, decreased myostatin, decreased SMAD 2/3, and elevated follistatin concentrations in human skeletal myoblast cultures with serum conditioned medium of patients in the acute phase of burn injury and conversely a reversed situation in patients in the chronic phase of burn injury. Thus, there is a biphasic response to burn trauma, initiated by an anabolic state and followed by long-term hypercatabolism. CONCLUSION: We conclude that the myostatin signaling pathway plays an important regulative role in burn-associated muscle wasting and that blockade of myostatin could prove to be a valuable treatment approach improving the rehabilitation process, QoL, and long-term survival after severe burn injury.

Prognostic Impact of Canonical TGF-ß Signaling in Urothelial Bladder Cancer.

Background and objectives: Dysregulation of TGF-ß signaling plays multiple roles in cancer development and progression. In the canonical TGF-ß pathway, TGF-ß regulates the expression of hundreds of target genes via interaction with Smads, signal transducers and transcriptional modulators. We evaluated the association of TGF-ß1, Smad2, and Smad4, the key components of canonical TGFß pathway, with clinicopathologic characteristics of urothelial bladder cancer, and assessed their prognostic value in prediction of patients' outcome. Materials and Methods: Immunohistochemical analysis of TGF-ß1, Smad2, and Smad4 expression was performed on 404 urothelial bladder cancer samples, incorporated in tissue microarrays. Expression status was correlated with clinicopathological and follow-up data. The median follow-up was 61 months. Results: High expression of TGF-ß1, Smad2, and Smad4 was detected in 68.1%, 31.7% and 45.2% of the tumors, respectively. TGF-ß1 overexpression was significantly associated with high tumor grade, and advanced pathologic stage (p < 0.001, respectively). Conversely, high Smad2 and Smad4 expression was linked to low tumor grade (p = 0,003, p = 0.048, respectively), and low tumor stage (p < 0.001, p = 0.003, respectively). Smad2 showed an inverse correlation with variant morphology and divergent differentiation of urothelial tumors (p = 0.014). High TGF-ß1 correlated directly, while Smad2 and Smad4 correlated inversely to cancer-specific death (p = 0.043, p = 0.003, and p = 0.022, respectively). There was a strong relationship between Smad2 and Smad4 expression (p < 0.001). Survival analyses showed that high Smad2 and Smad4 expression was associated with longer overall survival (p = 0.003, p = 0.034, respectively), while in multivariate regression analysis TGF-ß1 manifested as an independent predictor of poor outcome. Conclusions: Unraveling the complex roles and significance of TGF-ß signaling in urothelial bladder cancer might have important implications for therapy of this disease. Assessment of TGF-ß pathway status in patients with urothelial bladder cancer may provide useful prognostic information, and identify patients that could have the most benefit from therapy targeting TGF-ß signaling cascade.

Serine threonine kinase receptor associated protein regulates early follicle development in the mouse ovary.

The molecular mechanisms involved in regulating the development of small, gonadotrophin-independent follicles are poorly understood; however, many studies have highlighted an essential role for TGFB ligands. Canonical TGFB signalling is dependent upon intracellular SMAD proteins that regulate transcription. STRAP has been identified in other tissues as an inhibitor of the TGFB-SMAD signalling pathway. Therefore, in this study we aimed to determine the expression and role of STRAP in the context of early follicle development. Using qPCR, Strap, Smad3 and Smad7 revealed similar expression profiles in immature ovaries from mice aged 4-16 days containing different populations of early growing follicles. STRAP and SMAD2/3 proteins co-localised in granulosa cells of small follicles using immunofluorescence. Using an established culture model, neonatal mouse ovary fragments with a high density of small non-growing follicles were used to examine the effects of Strap knockdown using siRNA and STRAP protein inhibition by immuno-neutralisation. Both interventions caused a reduction in the proportion of small, non-growing follicles and an increase in the proportion and size of growing follicles in comparison to untreated controls, suggesting inhibition of STRAP facilitates follicle activation. Recombinant STRAP protein had no effect on small, non-growing follicles, but increased the mean oocyte size of growing follicles in the neonatal ovary model and also promoted the growth of isolated preantral follicles in vitro Overall findings indicate STRAP is expressed in the mouse ovary and is capable of regulating development of small follicles in a stage-dependent manner.

Increased Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates in a mouse model of aortic valve stenosis: Evidence of systemic activation of platelet-derived TGF-ß1 and correlation with cardiac dysfunction.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) has been implicated in the pathogenesis of aortic valve stenosis (AS). There is, however, little direct evidence for a role of active TGF-ß1 in AS due to the sensitivity of current assays. We searched for evidence of plasma TGF-ß1 activation by assaying Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates (PLAs) in a mouse model of AS (Reversa). METHODS: Echocardiography was used to measure AS and cardiac function. Intracellular phospho-flow cytometry in combination with optical fluorescence microscopy was used to detect PLAs and p-Smad2/3 levels. RESULTS: Reversa mice on a western diet developed AS, had significantly increased numbers of PLAs and more intense staining for p-Smad2/3 in both PLAs and single leukocytes (all p<0.05). p-Smad2/3 staining was more intense in PLAs than in single leukocytes in both diet groups (p<0.05) and correlated with plasma total TGF-ß1 levels (r=0.38, p=0.05 for PLAs and r=0.37, p=0.06 for single leukocytes) and reductions in ejection fraction (r=-0.42, p=0.03 for PLAs and r=-0.37, p=0.06 for single leukocytes). CONCLUSIONS: p-Smad2/3 staining is more intense in leukocytes of hypercholesterolemic mice that developed AS, suggesting increased circulating active TGF-ß1 levels. Leukocyte p-Smad2/3 may be a valuable surrogate indicator of circulating active TGF-ß1.

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

UNLABELLED: The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. CONTROL: 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle.

FGF signalling inhibits neural induction in human embryonic stem cells.

Human embryonic stem cells (hESCs) can exit the self-renewal programme, through the action of signalling molecules, at any given time and differentiate along the three germ layer lineages. We have systematically investigated the specific roles of three signalling pathways, TGFß/SMAD2, BMP/SMAD1, and FGF/ERK, in promoting the transition of hESCs into the neuroectoderm lineage. In this context, inhibition of SMAD2 and ERK signalling served to cooperatively promote exit from hESC self-renewal through the rapid downregulation of NANOG and OCT4. In contrast, inhibition of SMAD1 signalling acted to maintain SOX2 expression and prevent non-neural differentiation via HAND1. Inhibition of FGF/ERK upregulated OTX2 that subsequently induced the neuroectodermal fate determinant PAX6, revealing a novel role for FGF2 in indirectly repressing PAX6 in hESCs. Combined inhibition of the three pathways hence resulted in highly efficient neuroectoderm formation within 4 days, and subsequently, FGF/ERK inhibition promoted rapid differentiation into peripheral neurons. Our study assigns a novel, biphasic role to FGF/ERK signalling in the neural induction of hESCs, which may also have utility for applications requiring the rapid and efficient generation of peripheral neurons.

Novel Smad proteins localize to IR-induced double-strand breaks: interplay between TGFß and ATM pathways.

Cellular damage from ionizing radiation (IR) is in part due to DNA damage and reactive oxygen species, which activate DNA damage response (DDR) and cytokine signaling pathways, including the ataxia telangiectasia mutated (ATM) and transforming growth factor (TGF)ß/Smad pathways. Using classic double-strand breaks (DSBs) markers, we studied the roles of Smad proteins in DDR and the crosstalk between TGFß and ATM pathways. We observed co-localization of phospho-Smad2 (pSmad2) and Smad7 with DSB repair proteins following low and high linear energy transfer (LET) radiation in human fibroblasts and epithelial cells. The decays of both foci were similar to that of γH2AX foci. Irradiation with high LET particles induced pSmad2 and Smad7 foci tracks indicating the particle trajectory through cells. pSmad2 foci were absent in S phase cells, while Smad7 foci were present in all phases of cell cycle. pSmad2 (but not Smad7) foci were completely abolished when ATM was depleted or inactivated. In contrast, a TGFß receptor 1 (TGFßR1) inhibitor abrogated Smad7, but not pSmad2 foci at DSBs sites. In summary, we suggest that Smad2 and Smad7 contribute to IR-induced DSB signaling in an ATM or TGFßR1-dependent manner, respectively.

Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

Overexpression of Smad2 inhibits proliferation of gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Spatiotemporal inhibition of apical migration and proliferation of gingival epithelium are significant factors involved in periodontal regeneration. Transforming growth factor ß (TGF-ß) is important in multiple aspects of wound healing, and Smad2, a downstream transcription factor of TGF-ß, has an inhibitory effect on re-epithelialization during gingival wound healing. Therefore, we investigated the effects on migration and proliferation status, and intra/extracellular signaling regulated by Smad2 overexpression in gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells were isolated from the palatal gingival tissue of transgenic mice overexpressing Smad2 driven by the Keratin14 promoter. Smad2 expression was identified by western blotting and immunofluorescence analysis. Scratch assay and 5-bromo-2'-deoxyuridine staining were performed to assess cell migration and proliferation. To inactivate TGF-ß type I receptor, the cultures were supplemented with SB431542. Secreted TGF-ß was quantified by ELISA. Smad2 target gene expression was examined by real-time RT-PCR and in vivo immunofluorescence analysis of gingival junctional epithelium. RESULTS: Smad2-overexpressing cells were confirmed to have significant phosphorylated Smad2 in the nucleus. Scratch assay and 5-bromo-2'-deoxyuridine staining indicated that Smad2-overexpressing cells showed no significant differences in migration, but had reduced proliferation rates compared to wild-type controls. SB431542 significantly inhibited Smad2 phosphorylation, which coincided with restoration of the proliferation rate in Smad2-overexpressing cells. ELISA of TGF-ß release did not show any differences between genotypes. The cell cycle inhibitors, p15 and p21, showed significant upregulation in Smad2-overexpressing cells compared to wild-type controls. Moreover, junctional epithelium of the transgenic mice showed increased expression of P-Smad2, p15 and p21. CONCLUSION: The signaling activation triggered by overexpression of Smad2 was dependent on TGF-ß type I receptor, and the activated Smad2 increased p15 and p21 expression, responsible for inhibiting cell cycle entry, resulting in antiproliferative effects on gingival epithelial cells. Understanding of Smad2-induced signaling would be useful for possible clinical application to regulate gingival epithelial downgrowth.

Immunohistochemical localization of connective tissue growth factor, transforming growth factor-beta1 and phosphorylated-smad2/3 in the developing periodontium of rats.

BACKGROUND AND OBJECTIVE: Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta1 (TGF-ß1), and TGF-ß1-induced CTGF expression is regulated through the SMAD pathway. CTGF is implicated in the development of cartilage, bone and tooth. However, its expression in the developing periodontium is unclear. Therefore, we aimed to investigate the immunolocalization of CTGF, TGF-ß1 and phosphorylated SMAD2/3 (pSMAD2/3) in the developing periodontium of rats. MATERIAL AND METHODS: The maxillaries of Wistar rats, 2, 3, 7 and 12 wk of age, were used and the localization of CTGF, TGF-ß1 and pSMAD2/3 was detected using immunoperoxidase techniques. RESULTS: Hertwig' s epithelial root sheath (HERS) cells were strongly positive for CTGF and TGF-ß1, but not for pSMAD2/3. Positive staining for CTGF, TGF-ß1 and pSMAD2/3 was found in bone and periodontal ligament. In cementum, most cementoblasts associated with cellular cementum and some cementocytes stained strongly for CTGF, whereas cementoblasts associated with acellular cementum did not express CTGF. No signal for TGF-ß1 was observed in cellular and acellular cementum. In addition, most cementocytes were strongly positive for pSMAD2/3. CONCLUSION: CTGF, TGF-ß1 and pSMAD2/3 are localized in bone and periodontal ligament, but are differentially expressed in HERS and cementum. The results of our study indicate that the regulation of CTGF expression by TGF-ß1 might be cell-type specific in periodontium.

TGF-ß1, Smad-2/-3, Smad-1/-5/-8, and Smad-4 signaling factors are expressed in ameloblastomas, adenomatoid odontogenic tumors, and calcifying cystic odontogenic tumors: an immunohistochemical study.

OBJECTIVES: The TGF-ß/Smad signaling pathway regulates diverse cellular functions, including tooth development, and is involved in numerous pathological processes such as tumorigenesis. The aim of this study was to investigate the immunoexpression of the TGF-ß/Smad signaling pathway members in ameloblastoma (AM), calcifying cystic odontogenic tumor (CCOT), and adenomatoid odontogenic tumor (AOT). MATERIALS AND METHODS: This retrospective cross-sectional study included 65 tissue specimens: 34 AMs, 13 CCOTs, and 18 AOTs. Serial sections were immunohistochemically stained with TGF-ß1, Smad-4, Smad-1/-5/-8, and Smad-2/-3 antibodies, and a semiquantitative measurement of the positive cells was carried out by two oral pathologists using a 0-3 scale (0: no immunoreactivity, 1: <20% positive cells, 2: 20-50% positive cells, 3: >50% positive cells). RESULTS: All biomarkers studied were found significantly decreased in AM compared to CCOT and AOT. AOT and CCOT expressed Smad-1/-5/-8 more strongly compared to AM (OR = 11.66, P < 0.001 and OR = 5.34, P = 0.013, respectively), and Smad-2/-3 immunostaining was found significantly increased in CCOT (OR = 10.42, P = 0.001) and AOT (OR = 5.16, P < 0.004) compared to AM. Similarly, Smad-4 was expressed more strongly in AOT and CCOT compared to AM (P = 0.001), while AOT demonstrated a fivefold higher chance to express TGF-ß1 compared to AM (P = 0.011). CONCLUSION: TGF-ß/Smad signaling pathway is activated in AM, AOT, and CCOT. The statistically significant reduced TGF-ß1/Smad immunoexpression in AM compared to AOT/CCOT could be associated with the more aggressive biological behavior of AM including increased cell proliferation and reduced apoptosis and differentiation. Thus, the biomarkers TGF-ß, Smad-4, Smad-1/-5/-8, and Smad-2/-3 could serve as supplementary diagnostic indices between odontogenic tumors of high and low neoplastic dynamics.

EGFR signaling promotes TGFß-dependent renal fibrosis.

The mechanisms by which angiotensin II (Ang II) promotes renal fibrosis remain incompletely understood. Ang II both stimulates TGFß signaling and activates the EGF receptor (EGFR), but the relative contribution of these pathways to renal fibrogenesis is unknown. Using a murine model with EGFR-deficient proximal tubules, we demonstrate that upstream activation of EGFR-dependent ERK signaling is critical for mediating sustained TGFß expression in renal fibrosis. Persistent activation of the Ang II receptor stimulated ROS-dependent phosphorylation of Src, leading to sustained EGFR-dependent signaling for TGFß expression. Either genetic or pharmacologic inhibition of EGFR significantly decreased TGFß-mediated fibrogenesis. We conclude that TGFß-mediated tissue fibrosis relies on a persistent feed-forward mechanism of EGFR/ERK activation through an unexpected signaling pathway, highlighting EGFR as a potential therapeutic target for modulating tissue fibrogenesis.

Seasonal changes in immunoreactivity of activin signaling component proteins in wild ground squirrel testes.

The seasonal spermatogenesis and localization of inhibin/activin subunits (alpha, betaA, betaB) in the testes of wild ground squirrel has been previously described; however, the expression pattern of activin receptors and cytoplasmic signaling SMADs has not been detected in any seasonal breeders. The objective of this study was to investigate the abundance and cellular localization of activin signaling components in testes of the wild ground squirrel during the breeding and nonbreeding seasons. The immunolocalizations of ActRIIB (activin type II receptor B) and activin-related SMADs (phospho-SMAD2/3, SMAD4 and SMAD7) were observed by immunohistochemistry. Total proteins were extracted from testicular tissues in the breeding and nonbreeding seasons and were used for Western blotting analysis for ActRIIB and SMADs. Immunoreactivities of activin signaling components were greater in the testes of the breeding season, and then decreased to a relatively low level in the nonbreeding season. ActRIIB and related SMADs were widely spread in the active testes, while spermatogonia were the predominant cellular sites of activin signal transduction during arrested spermatogenesis. The dynamic regulation of activin type II receptor and SMADs indicated that the activin signal pathway played an important paracrine role in seasonal spermatogenesis of the wild ground squirrel. Furthermore, the distinct localizations and immunoreactivity of ActRIIB and SMADs might suggest different functions of activin in seasonal spermatogenesis.

Transforming growth factor ß repressor, SnoN, is overexpressed in human gastrointestinal stromal tumors.

PURPOSE: The transforming growth factor bgr; (TGF-ß)/ Smad pathway is implicated in the development of interstitial cells of Cajal. The aim of this study was to examine the role of this pathway in human gastrointestinal stromal tumors (GISTs). METHODS: The expression of TGF-ß receptor II (TßRII), phosphorylated Smad2 (p-Smad2), SnoN, p21(WAF17sol;CIP1) and p27(KIP1) was examined by immunohistochemistry in 30 hu-man GISTs in relation to prognostic factors. RESULTS: TßRII was expressed in 76.9% of the cases. All cases were positive for p-Smad2 and SnoN, with significantly higher expression levels in small intestinal compared to gastric GISTs. Downregulation of p21(WAF1/CIP1) and p27(KIP1) was found in 78.6% and 46.4% of the cases respectively, while cytoplasmic expression of p27(KIP1) was also noted in 50% of GISTs. CONCLUSIONS: TGF-ß/Smad pathway may contribute to GIST pathogenesis. SnoN overexpression and low levels of p21(WAF1)/CIP1 and p27(KIP1) may be of importance in GISTs.

Increased cavernous expression of transforming growth factor-ß1 and activation of the Smad signaling pathway affects erectile dysfunction in men with spinal cord injury.

INTRODUCTION: Transforming growth factor-ß1 (TGF-ß1) is implicated in bladder fibrosis after spinal cord injury (SCI) and in the fibrosis in the corpus cavernosum tissue after cavernous nerve injury. AIM: We investigated the differential expression of TGF-ß1 and the Smad transcription factor, the key molecule for the initiation of TGF-ß-mediated fibrosis, in cavernous tissue from SCI patients. METHODS: After obtaining informed consent and approval from the patients and our institutional review board, we enrolled 5 patients with psychogenic erectile dysfunction (ED) (mean age 36.8 years; range 20-50 years) and 10 patients with neurogenic ED from SCI (mean age 38.8 years; range 18-50 years). Cavernous tissues were obtained by percutaneous biopsy and stained with Masson trichrome, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL), or antibodies to TGF-ß1 and phospho-Smad2. MAIN OUTCOME MEASURES: Semi-quantitative analysis of TGF-ß1 and phospho-Smad2 was performed, and the numbers of apoptotic cells were counted. We also quantified the cavernous collagen area with the use of an image analyzer system. RESULTS: The expression of TGF-ß1 and phospho-Smad2 protein was significantly higher in the SCI group than in the psychogenic group. The TUNEL assay revealed a higher apoptotic index in the SCI group than in the psychogenic group. Higher TGF-ß1 and phospho-Smad2 expression and more apoptotic cells were noted mainly in endothelial cells, smooth muscle cells, and fibroblasts of the SCI group. Double labeling of cavernous tissue with TUNEL and antibody to phospho-Smad2 revealed that most TUNEL-positive cells showed immunoreactivity to phospho-Smad2 staining. Cavernous collagen content was significantly greater in the SCI group than in the psychogenic group. CONCLUSION: Upregulation of TGF-ß1 and activation of the Smad signaling pathway may play important roles in SCI-induced cavernous fibrosis and deterioration of erectile function, which warrants early pharmacological intervention to protect erectile tissue from irreversible damage.

Prognostic factors for idiopathic pulmonary fibrosis: clinical, physiologic, pathologic, and molecular aspects.

BACKGROUND: Previous studies identified clinical and physiologic factors of idiopathic pulmonary fibrosis (IPF) that are related to an increased risk of mortality. But there are few studies about histologic and molecular approach. OBJECTIVE: We investigated whether the C-reactive protein (CRP), fibroblastic foci, phosphorylated Smad2/3 (p-Smad2/3), tumor growth factor-beta (TGF-beta), TGF-beta receptor II (TbetaRII), and the polymorphism of the TGF-beta1 codon 10 are associated with the progression of IPF patients. DESIGN: Eighty-six IPF patients who underwent surgical lung biopsies were examined. For each patient, clinical and physiologic parameters were investigated, and we performed immunohistochemical staining for p-Smad2/3 and TbetaRII, and genotyping of the TGF-beta1 codon 10 polymorphism. RESULTS: Age at diagnosis, gender, symptom duration, and smoking status did not show a significant association. However, the amount of smoking (p = 0.002), severe reduction in the percentages of predicted forced vital capacity (p = 0.013) and diffusion lung capacity of carbon monoxide (p = 0.023), CRP (p = 0.009) at diagnosis, and fibroblastic foci (p = 0.026) were associated with a poor prognosis. Cellularity, fibrosis, expression level of p-Smad2/3 and TbetaRII, and genotype of the TGF-beta1 codon 10 polymorphism did not have a statistically significant association with the prognosis. CONCLUSION: This study confirmed the amount of smoking, abrupt decrease in follow-up pulmonary function parameters, fibroblastic foci, and increased levels of CRP concentration at diagnosis were significantly associated with poor survival. Larger studies are required to confirm all prognostic factors including CRP.

Transforming growth factor-ß signaling pathway in Marfan's syndrome: a preliminary histopathological study.

BACKGROUND: Marfan's syndrome is an inherited disorder that affects the connective tissue. It has been proposed that mutations of FBN1 gene or of transforming growth factor (TGF)-beta type II receptor may be responsible for its pathogenesis. However, the role of TGF-beta signaling pathway in the development of Marfan's syndrome has not been comprehensively investigated. MATERIALS AND METHODS: Surgical specimens of the aorta were obtained from two female Marfan patients, and the control aortic tissue was taken from an autopsy of a healthy individual. The aortic specimens were examined with hematoxylin-eosin, Masson's trichrome, von Gieson/victoria blue-van Gieson bichrome, and immunohistochemical stainings of TGF-beta1, TGF-beta type I receptor, Smad2/3, Smad4 and Smad7. RESULTS: Hematoxylin-eosin staining demonstrated severe elastic lamellar disruption and patchy vascular smooth muscle dissolution in the aortic media of the Marfan patients. Collagen deposition, interlamilar elastic fiber fragmentation, loss or proliferation, and acid mucopolysaccharide accumulation were observed in the disarrayed aortic wall structures of Marfan patients by Masson's trichrome, victoria blue-van Gieson bichrome, and Alcian blue and periodic schiff's (AB-PAS) stainings, respectively. By immunohistochemistry, structural disruptions with enhanced TGF-beta;1 in the cytoplasm, Smad2/3 in the interstices, Smad4 in the cytoplasm, nuclei or interstices, and OOO Smad7, in the nucleus along with attenuated TGF-beta type I receptor in the aortic tissues of Marfan patients in comparison to the healthy control. CONCLUSIONS: Marfan patients may have aberrant TGF-beta signaling pathway associated with increased collagen deposition, interlamilar elastic fiber degenerative changes, and acid mucopolysaccharide accumulation. The signaling dysregulation may play an important role in the pathogenesis of this genetic disorder.

Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with peroxidase detection.

BACKGROUND: The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research. METHODS: We used horseradish peroxidase (HRP)-conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event. RESULTS: We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both in cultured cells and in tissue samples. CONCLUSIONS: The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.

Signal transduction and metabolism in chondrocytes is modulated by lactoferrin.

OBJECTIVE: Activation of granulocytes causes a considerable rise in the concentration of lactoferrin (Lf) in synovial fluid (SF). We here investigate consequences thereof on signal transduction and the balance between catabolic and anabolic metabolism in chondrocytes. METHODS: Signal transduction was analysed in cultured chondrocytes by immunodetection of mitogen activated protein kinases (MAPK) and analysis of Smad2 translocation to the nucleus. Expression levels of matrix metalloproteinases (MMPs) and of aggrecan were measured by reverse-transcription-PCR. The proteolytic activity of MMPs was ascertained by zymography. Expression of the low-density-lipoprotein-receptor-related-protein-1 (LRP-1), a Lf receptor for signalling, was assayed by immunohistochemistry in cartilage and in cultured chondrocytes by immunoblotting. RESULTS: We found LRP-1 expressed in dedifferentiated chondrocytes in culture and in cartilage tissue preferentially on the articular surface where it can encounter Lf within SF. Lf stimulated proliferation of chondrocytes, comparable to transforming growth factor-beta1 (TGFbeta1) and activated p38 and the extracellular-signal regulated-kinases 1/2 (ERK1/2) within minutes. Surprisingly, Lf induced nuclear Smad2 translocation, a signal pathway ascribed to TGFbeta receptor activation. Lf significantly increased the levels of catabolic indicators such as MMP1, MMP2, MMP3 and MMP13 and inhibited aggrecan synthesis. CONCLUSION: Lf is a robust regulator of chondrocyte metabolism, comparable to TGFbeta1. The catabolic influence together with the proliferative stimulus indicates a function as an early phase cytokine, enhancing MMPs, necessary for degradation of damaged tissue and stimulating proliferation of chondrocytes, necessary for reconstruction.