Endurance Training Provokes Arrhythmogenic Right Ventricular Cardiomyopathy Phenotype in Heterozygous Desmoglein-2 Mutants: Alleviation by Preload Reduction.

Desmoglein-2 mutations are detected in 5-10% of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). Endurance training accelerates the development of the ARVC phenotype, leading to earlier arrhythmic events. Homozygous Dsg2 mutant mice develop a severe ARVC-like phenotype. The phenotype of heterozygous mutant (Dsg2mt/wt) or haploinsufficient (Dsg20/wt) mice is still not well understood. To assess the effects of age and endurance swim training, we studied cardiac morphology and function in sedentary one-year-old Dsg2mt/wt and Dsg20/wt mice and in young Dsg2mt/wt mice exposed to endurance swim training. Cardiac structure was only occasionally affected in aged Dsg20/wt and Dsg2mt/wt mice manifesting as small fibrotic foci and displacement of Connexin 43. Endurance swim training increased the right ventricular (RV) diameter and decreased RV function in Dsg2mt/wt mice but not in wild types. Dsg2mt/wt hearts showed increased ventricular activation times and pacing-induced ventricular arrhythmia without obvious fibrosis or inflammation. Preload-reducing therapy during training prevented RV enlargement and alleviated the electrophysiological phenotype. Taken together, endurance swim training induced features of ARVC in young adult Dsg2mt/wt mice. Prolonged ventricular activation times in the hearts of trained Dsg2mt/wt mice are therefore a potential mechanism for increased arrhythmia risk. Preload-reducing therapy prevented training-induced ARVC phenotype pointing to beneficial treatment options in human patients.

The role of desmoglein-2 in kidney disease.

Desmosomes are multi-protein cell-cell adhesion structures supporting cell stability and mechanical stress resilience of tissues, best described in skin and heart. The kidney is exposed to various mechanical stimuli and stress, yet little is known about kidney desmosomes. In healthy kidneys, we found desmosomal proteins located at the apical-junctional complex in tubular epithelial cells. In four different animal models and patient biopsies with various kidney diseases, desmosomal components were significantly upregulated and partly miss-localized outside of the apical-junctional complexes along the whole lateral tubular epithelial cell membrane. The most upregulated component was desmoglein-2 (Dsg2). Mice with constitutive tubular epithelial cell-specific deletion of Dsg2 developed normally, and other desmosomal components were not altered in these mice. When challenged with different types of tubular epithelial cell injury (unilateral ureteral obstruction, ischemia-reperfusion, and 2,8-dihydroxyadenine crystal nephropathy), we found increased tubular epithelial cell apoptosis, proliferation, tubular atrophy, and inflammation compared to wild-type mice in all models and time points. In vitro, silencing DSG2 via siRNA weakened cell-cell adhesion in HK-2 cells and increased cell death. Thus, our data show a prominent upregulation of desmosomal components in tubular cells across species and diseases and suggest a protective role of Dsg2 against various injurious stimuli.

Desmoglein 2 regulates cardiogenesis by restricting hematopoiesis in the developing murine heart.

Cardiac morphogenesis relies on intricate intercellular signaling. Altered signaling impacts cardiac function and is detrimental to embryonic survival. Here we report an unexpected regulatory role of the desmosomal cell adhesion molecule desmoglein 2 (Dsg2) on murine heart development. A large percentage of Dsg2-mutant embryos develop pericardial hemorrhage. Lethal myocardial rupture is occasionally observed, which is not associated with loss of cardiomyocyte contact but with expansion of abnormal, non-myocyte cell clusters within the myocardial wall. Two types of abnormal cell clusters can be distinguished: Type A clusters involve endocard-associated, round-shaped CD31+ cells, which proliferate and invade the myocardium. They acquire Runx1- and CD44-positivity indicating a shift towards a hematopoietic phenotype. Type B clusters expand subepicardially and next to type A clusters. They consist primarily of Ter119+ erythroid cells with interspersed Runx1+/CD44+ cells suggesting that they originate from type A cell clusters. The observed pericardial hemorrhage is caused by migration of erythrocytes from type B clusters through the epicardium and rupture of the altered cardiac wall. Finally, evidence is presented that structural defects of Dsg2-depleted cardiomyocytes are primary to the observed pathogenesis. We propose that cardiomyocyte-driven paracrine signaling, which likely involves Notch1, directs subsequent trans-differentiation of endo- and epicardial cells. Together, our observations uncover a hitherto unknown regulatory role of Dsg2 in cardiogenesis.

Autophagy and Endoplasmic Reticulum Stress during Onset and Progression of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is a heritable, potentially lethal disease without a causal therapy. AC is characterized by focal cardiomyocyte death followed by inflammation and progressive formation of connective tissue. The pathomechanisms leading to structural disease onset and progression, however, are not fully elucidated. Recent studies revealed that dysregulation of autophagy and endoplasmic/sarcoplasmic reticulum (ER/SR) stress plays an important role in cardiac pathophysiology. We therefore examined the temporal and spatial expression patterns of autophagy and ER/SR stress indicators in murine AC models by qRT-PCR, immunohistochemistry, in situ hybridization and electron microscopy. Cardiomyocytes overexpressing the autophagy markers LC3 and SQSTM1/p62 and containing prominent autophagic vacuoles were detected next to regions of inflammation and fibrosis during onset and chronic disease progression. mRNAs of the ER stress markers Chop and sXbp1 were elevated in both ventricles at disease onset. During chronic disease progression Chop mRNA was upregulated in right ventricles. In addition, reduced Ryr2 mRNA expression together with often drastically enlarged ER/SR cisternae further indicated SR dysfunction during this disease phase. Our observations support the hypothesis that locally altered autophagy and enhanced ER/SR stress play a role in AC pathogenesis both at the onset and during chronic progression.

Inflammation shapes pathogenesis of murine arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is an incurable genetic disease, whose pathogenesis is poorly understood. AC is characterized by arrhythmia, fibrosis, and cardiodilation that may lead to sudden cardiac death or heart failure. To elucidate AC pathogenesis and to design possible treatment strategies of AC, multiple murine models have been established. Among them, mice carrying desmoglein 2 mutations are particularly valuable given the identification of desmoglein 2 mutations in human AC and the detection of desmoglein 2 auto-antibodies in AC patients. Using two mouse strains producing either a mutant desmoglein 2 or lacking desmoglein 2 in cardiomyocytes, we test the hypothesis that inflammation is a major component of disease pathogenesis. We show that multifocal cardiomyocyte necrosis initiates a neutrophil-dominated inflammatory response, which also involves macrophages and T cells. Increased expression of Ccl2/Ccr2, Ccl3/Ccr5, and Cxcl5/Cxcr2 mRNA reflects the observed immune cell recruitment. During the ensuing acute disease phase, Mmp12+ and Spp1+ macrophages and T cells accumulate in scars, which mature from cell- to collagen-rich. The expression of Cx3cl1/Cx3cr1, Ccl2/Ccr2, and Cxcl10/Cxcr3 dominates this disease phase. We furthermore find that during chronic disease progression macrophages and T cells persist within mature scars and are present in expanding interstitial fibrosis. Ccl12 and Cx3cl1 are predominant chemokines in this disease phase. Together, our observations provide strong evidence that specific immune cell populations and chemokine expression profiles modulate inflammatory and repair processes throughout AC progression.

Desmoglein 2 mutation provokes skeletal muscle actin expression and accumulation at intercalated discs in murine hearts.

Arrhythmogenic cardiomyopathy (AC) is an incurable progressive disease that is linked to mutations in genes coding for components of desmosomal adhesions that are localized to the intercalated disc region, which electromechanically couples adjacent cardiomyocytes. To date, the underlying molecular dysfunctions are not well characterized. In two murine AC models, we find an upregulation of the skeletal muscle actin gene (Acta1), which is known to be a compensatory reaction to compromised heart function. Expression of this gene is elevated prior to visible morphological alterations and clinical symptoms, and persists throughout pathogenesis with an additional major rise during the chronic disease stage. We provide evidence that the increased Acta1 transcription is initiated through nuclear activation of the serum response transcription factor (SRF) by its transcriptional co-activator megakaryoblastic leukemia 1 protein (MKL1, also known as MRTFA). Our data further suggest that perturbed desmosomal adhesion causes Acta1 overexpression during the early stages of the disease, which is amplified by transforming growth factor ß (TGFß) release from fibrotic lesions and surrounding cardiomyocytes during later disease stages. These observations highlight a hitherto unknown molecular AC pathomechanism.

Desmoglein 2, but not desmocollin 2, protects intestinal epithelia from injury.

Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell-cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2ΔIEC; DSC2ΔIEC). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2ΔIEC, but not DSC2ΔIEC mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2ΔIEC mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2ΔIEC animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2ΔIEC mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2ΔIEC mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.

Ultrastructural changes in endometrial desmosomes of desmoglein 2 mutant mice.

The intercellular binding of desmosomal junctions is mediated by cadherins of the desmoglein (Dsg) and desmocollin (Dsc) type. Dsg2 mutant mice with deletion of a substantial segment of the extracellular EC1-EC2 domain, which is believed to participate in homo- and heterophilic desmosomal cadherin interactions, develop cardiac fibrosis and ventricular dilation. Widening of the intercellular cleft and complete intercalated disc ruptures can be observed in the hearts of these mice. Since a reduced litter size of homozygous Dsg2 mutant mice was noted and a functional correlation between desmosomes and embryo implantation has been deduced from animal studies, we looked for an alteration of desmosomes in uterine endometrial epithelium. Shape and number of desmosomes as well as the expression of Dsg2 and the desmosomal plaque protein desmoplakin (Dsp) were investigated by electron microscopy and immunohistochemistry in 12 oestrous-dated mice (7 wild type and 5 homozygous Dsg2 mutant mice) at the age of 9-17 weeks. The immunohistochemical detection of Dsg2 was diminished in the mutants and the number of desmosomes was significantly reduced as revealed by electron microscopy. In addition, the intercellular desmosomal space measured in electron micrographs was considerably widened in the Dsg2 mutants. The increased intercellular spacing can be explained by the partial deletion of the extracellular EC1-EC2 domain of Dsg2. Whether these changes explain the reduced number of offspring of homozygous Dsg2 mutant mice remains to be further investigated.

Cardiomyocyte Hypertrophy in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is a hereditary disease leading to sudden cardiac death or heart failure. AC pathology is characterized by cardiomyocyte loss and replacement fibrosis. Our goal was to determine whether cardiomyocytes respond to AC progression by pathological hypertrophy. To this end, we examined tissue samples from AC patients with end-stage heart failure and tissue samples that were collected at different disease stages from desmoglein 2-mutant mice, a well characterized AC model. We find that cardiomyocyte diameters are significantly increased in right ventricles of AC patients. Increased mRNA expression of the cardiac stress marker natriuretic peptide B is also observed in the right ventricle of AC patients. Elevated myosin heavy chain 7 mRNA expression is detected in left ventricles. In desmoglein 2-mutant mice, cardiomyocyte diameters are normal during the concealed disease phase but increase significantly after acute disease onset on cardiomyocyte death and fibrotic myocardial remodeling. Hypertrophy progresses further during the chronic disease stage. In parallel, mRNA expression of myosin heavy chain 7 and natriuretic peptide B is up-regulated in both ventricles with right ventricular preference. Calcineurin/nuclear factor of activated T cells (Nfat) signaling, which is linked to pathological hypertrophy, is observed during AC progression, as evidenced by Nfatc2 and Nfatc3 mRNA in cardiomyocytes and increased mRNA of the Nfat target regulator of calcineurin 1. Taken together, we demonstrate that pathological hypertrophy occurs in AC and is secondary to cardiomyocyte loss and cardiac remodeling.

Loss of plakoglobin immunoreactivity in intercalated discs in arrhythmogenic right ventricular cardiomyopathy: protein mislocalization versus epitope masking.

AIMS: To examine the relevance and cause of reduced plakoglobin IF in intercalated discs for arrhythmogenic right ventricular cardiomyopathy (ARVC) and ARVC-like disease in mouse and human. METHODS AND RESULTS: Normalized semi-quantitative IF measurements were performed in a standardized format in desmoglein 2-mutant mice with an ARVC-like phenotype (n = 6) and in cardiac biopsies from humans with ARVC and non-ARVC heart disease (n = 10). Reduced plakoglobin staining was detectable in ARVC only with one antibody directed against a defined epitope but not with three other antibodies reacting with different epitopes of plakoglobin. CONCLUSIONS: Reduced plakoglobin staining in intercalated discs of heart tissue from human ARVC patients and in a murine ARVC model is caused by alterations in epitope accessibility and not by protein relocalization.

Desmoglein 2-Dependent Arrhythmogenic Cardiomyopathy Is Caused by a Loss of Adhesive Function.

BACKGROUND: The desmosomal cadherin desmoglein 2 (Dsg2) localizes to the intercalated disc coupling adjacent cardiomyocytes. Desmoglein 2 gene (DSG2) mutations cause arrhythmogenic cardiomyopathy (AC) in human and transgenic mice. AC is characterized by arrhythmia, cardiodilation, cardiomyocyte necrosis with replacement fibrosis, interstitial fibrosis, and intercalated disc dissociation. The genetic DSG2 constellations encountered are compatible with loss of adhesion and altered signaling. To further elucidate pathomechanisms, we examined whether heart-specific Dsg2 depletion triggers cardiomyopathy. METHODS AND RESULTS: Because DSG2 knockouts die during early embryogenesis, mice were prepared with cardiomyocyte-specific DSG2 ablation. Healthy transgenic animals were born with a functional heart presenting intercalated discs with incorporated desmosomal proteins. Dsg2 protein expression was reduced below 3% in the heart. All animals developed AC during postnatal growth with pronounced chamber dilation, calcifying cardiomyocyte necrosis, aseptic inflammation, interstitial and focal replacement fibrosis, and conduction defects with altered connexin 43 distribution. Electron microscopy revealed absence of desmosome-like structures and regional loss of intercalated disc adhesion. Mice carrying 2 mutant DSG2 alleles coding for Dsg2 lacking part of the adhesive EC1-EC2 domains present an indistinguishable phenotype, which is similar to that observed in human AC patients. CONCLUSIONS: The observations show that the presence of Dsg2 is not essential for late heart morphogenesis and for cardiac contractility to support postnatal life. On increasing mechanical demands, heart function is severely compromised as evidenced by the onset of cardiomyopathy with pronounced morphological alterations. We propose that loss of Dsg2 compromises adhesion, and that this is a major pathogenic mechanism in DSG2-related and probably other desmosome-related ACs.

Mammaglobin 1: not only a breast-specific and tumour-specific marker, but also a hormone-responsive endometrial protein.

AIMS: The secretoglobin mammaglobin 1 (MGB1) is strongly expressed in breast tumours, and is therefore used to detect breast cancer metastases, although it has also been detected in other tissues. The aim of this study was to examine MGB1 expression and its hormonal regulation in human endometrium to further investigate the use of MGB1 as a marker molecule. METHODS AND RESULTS: Mammaglobin 1 expression was assessed immunohistochemically in endometrial samples from 60 normal fertile patients throughout the menstrual cycle, in 49 endometriotic tissue samples, in 15 endometrial adenocarcinomas, and in 36 breast carcinomas. In addition, 25 endometrial samples were analysed by western blot and quantitative real-time reverse transcription polymerase chain reaction. To prove hormonal regulation, primary endometrial epithelial cells were cultured with 17ß-oestradiol and promegestone. MGB1 was detected in human endometrial tissue, with peak expression during the luteal phase, in 31% of endometriotic samples, in 53% of endometrial adenocarcinomas, and in 64% of breast carcinomas. MGB1 mRNA expression was increased in vitro by hormonal treatment. CONCLUSIONS: Our data show that MGB1 expression is not restricted to normal and malignant breast tissue. Besides its documented occurrence in endometriotic and malignant endometrial tissues, MGB1 is also expressed in normal human endometrium, and such expression is controlled by steroid hormones.

Histological and ultrastructural abnormalities in murine desmoglein 2-mutant hearts.

Mice carrying a deletion of the adhesive extracellular domain of the desmosomal cadherin desmoglein 2 develop an arrhythmogenic right ventricular cardiomyopathylike phenotype with ventricular dilation, fibrosis and arrhythmia. To unravel the sequence of myocardial alterations and to identify potential pathomechanisms, histological analyses were performed on mutant hearts from the juvenile to the adult state, i.e., between 2 and 13 weeks. At an age of 2 weeks 30% of mutants presented lesions,which were visible as white plaques on the heart surface or in the septum. From 4 weeks onwards, all mutants displayed a cardiac phenotype. Dying cardiomyocytes with calcification were found in lesions of all ages. But lesions of young mutant animals contained high amounts of CD45+ immune cells and little collagen fibers, whereas lesions of the older animals were collagen-rich and harbored only a small but still significantly increased number of CD45+ cells. Electron microscopy further showed that distinct desmosomes cannot be distinguished in intercalated discs of mutant hearts. Widening of the intercellular cleft and even complete dissociation of intercalated discs were often observed close to lesions. Disturbed sarcomer structure, altered Z-discs, multiple autophagic vacuoles and swollen mitochondria were other prominent pathological features. Taken together, the following scenario is suggested: mutant desmoglein 2 cannot fully support the increased mechanical requirements placed on intercalated disc adhesion during postnatal heart development, resulting in compromised adhesion and cell stress. This induces cardiomyocyte death, aseptic inflammation and fibrotic replacement. The acute stage of scar formation is followed by permanent impairment of the cardiac function.

Comparative morphological and immunohistochemical study of human meningioma after intracranial transplantation into nude mice.

Although surgical resection of benign human meningiomas is the primary goal, in case of relapse or when they are not fully resectable, other strategies including chemotherapeutical treatment would be appropriate. The initial evaluation of chemotherapeutical agents requires an appropriate tumor model, where the natural characteristics of the original benign tumor is reflected. We here tested, whether primary cell cultures of benign human meningiomas would reliably grow after intracranial transplantation into mice, and whether they would show histomorphological and immunohistochemical characteristics of the original human tumor. Cells of 11 benign human meningiomas were transplanted into the prefrontal cortex of nude mice. After 3 months, the mice were sacrificed and their brains were histologically processed for morphological characterization and measurement of tumor volume. Additionally, the proliferation index (PI), the microvessel density, and epithelial membrane antigen (EMA) were compared between human meningiomas and tumors grown in mice by using immunohistochemical methods. Further, cyclooxygenase-2 (COX-2) expression, a possible target for pharmacological manipulation, was examined. The results showed in almost all mice (93%) a tumor formation with meningothelial histomorphology comparable to the original human tumors. The PI, vascular density and COX-2 expression were similar between human and mice meningiomas, but EMA expression was reduced in mice (P<0.01). In conclusion an implantation of benign human meningioma primary cell cultures in mice reliably results in tumor formation with morphological and immunohistological features comparable to the original human tumor. This model may therefore be suitable to test novel therapeutic agents.

Desmoglein 2 mutant mice develop cardiac fibrosis and dilation.

Desmosomes are cell-cell adhesion sites and part of the intercalated discs, which couple adjacent cardiomyocytes. The connection is formed by the extracellular domains of desmosomal cadherins that are also linked to the cytoskeleton on the cytoplasmic side. To examine the contribution of the desmosomal cadherin desmoglein 2 to cardiomyocyte adhesion and cardiac function, mutant mice were prepared lacking a part of the extracellular adhesive domain of desmoglein 2. Most live born mutant mice presented normal overall cardiac morphology at 2 weeks. Some animals, however, displayed extensive fibrotic lesions. Later on, mutants developed ventricular dilation leading to cardiac insufficiency and eventually premature death. Upon histological examination, cardiomyocyte death by calcifying necrosis and replacement by fibrous tissue were observed. Fibrotic lesions were highly proliferative in 2-week-old mutants, whereas the fibrotic lesions of older mutants showed little proliferation indicating the completion of local muscle replacement by scar tissue. Disease progression correlated with increased mRNA expression of c-myc, ANF, BNF, CTGF and GDF15, which are markers for cardiac stress, remodeling and heart failure. Taken together, the desmoglein 2-mutant mice display features of dilative cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy, an inherited human heart disease with pronounced fibrosis and ventricular arrhythmias that has been linked to mutations in desmosomal proteins including desmoglein 2.

Cyclooxygenase-2 supports tumor proliferation in vestibular schwannomas.

BACKGROUND: Recent studies have shown that cyclooxygenase-2 (COX-2) plays an important role in tumor growth and neovascularization. However, COX-2 expression in vestibular schwannomas (VSs) has not been investigated. OBJECTIVE: To analyze the pattern of COX-2 expression in sporadic and neurofibromatosis type 2 (NF2)-associated VSs and its relationship with tumor proliferation and microvessel density. METHODS: Fifteen sporadic and 15 NF2-associated VSs were examined for COX-2 expression, microvessel density, and proliferation rate by immunohistochemical methods. Immunohistochemical scores were used to interpret the extent and intensity of COX-2 staining. Microvessel density (MVD) was determined using von Willebrand factor (vWf). Proliferation rate was quantified using Ki-67. The relationship among COX-2 expression, MVD, and proliferation rate was statistically analyzed. RESULTS: COX-2 expression was detected in 29 (96.67%) of 30 VSs, with no significant difference between sporadic and NF2-associated VSs (P = .722). In 6 (20%) VSs, COX-2 expression was graded as strong, in 12 (40%) as moderate, and in 11 (36.7%) as weak. VSs with high proliferation showed significantly higher COX-2 expression (P = .015) than VSs with low proliferation. COX-2 expression and MVD did not show specific biological correlations (P = .035). CONCLUSION: Our data demonstrate that COX-2 is expressed in VSs. High COX-2 expression in VSs with high proliferation rates suggests that the COX-2 pathway may be involved in the development and growth of VSs.

Dendritic cells are equally distributed in intrauterine and tubal ectopic pregnancies.

OBJECTIVE: To examine different stages of dendritic cells (DCs) in intrauterine (IUPs) and viable tubal (VTPs) pregnancies to further elucidate mechanisms of fetomaternal tolerance and extravillous trophoblast invasion. DESIGN: Experimental study on patient-controlled material. SETTING: University hospital. PATIENT(S): Seven women with normal IUPs and ten with VTPs in the first trimester. INTERVENTION(S): Suction curettage in IUP, laparoscopy in VTP. MAIN OUTCOME MEASURE(S): Immunohistochemistry for cytokeratin-7 (trophoblast), CD83 (mature DCs), DEC205 (activated but not fully mature DCs), DC-SIGN (immature macrophage-like DCs), and CD14 (macrophages) alone and in double staining. RESULT(S): The numbers of CD83+ and DEC205+ cells were similarly low in IUP and VTP (0.83 and 0.44 cells/mm2; 2.28 and 2.96 cells/mm2). The number of DC-SIGN+ cells was higher, though without significant differences among the entities examined (57.5 and 47.4 cells/mm2). About two-thirds of DC-SIGN+ cells were also CD14+ in IUP and VTP. CONCLUSION(S): The almost equal distribution of CD83+, DEC205+, and DC-SIGN+ cells in IUP and VTP suggests analogue control mechanisms in intrauterine and extrauterine DC differentiation and a comparable role of these DCs for the development of fetomaternal tolerance.

Implementation of External Quality Assurance Trials for Immunohistochemically Determined Breast Cancer Biomarkers in Germany.

Besides typing and grading of breast cancer, Pathologists are involved in the determination of biomarkers, such as steroid hormone receptors and HER2, which are of utmost importance in adjuvant therapy. There have been concerns with regard to security and reproducibility of the biomarker assays done on tissue sections applying either immunohistochemistry or in-situ hybridisation. In order to assure the quality of these biomarker assays, a number of measures are required, among them external proficiency testing. Therefore, external quality assurance trials have been implemented in Germany. In the period of 2002-2007, 5 consecutive trials were conducted with up to 180 participating laboratories. Tissue microarrays with 20-24 different breast cancer samples including cell lines enabled that a huge number of pathologists were challenged with identical samples which provides the prerequisite for comparability. Because there is no legal duress to undergo external proficiency testing in histopathology, all laboratories that took part volunteered to do so. These innovative quality assurance trials (Qualitätsinitiative Pathologie, QuIP) will be continued in the future on an annual or bi-annual basis. Participation is recommended for pathology departments involved in the service for breast units. The organisational frame work of the trials is described here.

Class I histone deacetylase expression in the human cyclic endometrium and endometrial adenocarcinomas.

BACKGROUND: Class I histone deacetylases (HDACs) and acetylases (HATs) are members of transcriptional pre-initiation complexes assembled by steroid hormone receptors. Recently, HDAC inhibitors were shown to enhance differentiation of endometrial fibroblasts and endometrial adenocarcinomas. However, there is only rare information on HDAC and HAT expression in the human endometrium. METHODS: HDAC-1, -2, -3 and HAT (PCAF and GCN5) mRNA expression was studied in tissue from premenopausal women undergoing hysterectomy by real-time or semiquantitative RT-PCR. HDAC protein expression was assessed by Western Blot and immunohistochemistry. In endometrial adenocarcinomas (n = 17), HDAC-1 expression was studied by immunohistochemistry. RESULTS: In the human endometrium, HDAC-1, -2, -3 and PCAF mRNA are expressed without cyclical changes. Western blot analysis demonstrated that HDAC-2 protein expression was slightly, but significantly elevated in the secretory phase (P < 0.01 versus day 5-8), whereas HDAC-1 and -3 protein expression was constitutive throughout the menstrual cycle. By immunohistochemistry, nuclear expression of HDAC proteins was detected in all endometrial cell types. In the case of HDAC-3, immunostaining was significantly reduced in the endometrial surface epithelium on day 6-10 (P < 0.01 versus days 15-18 and 24-28). Compared to normal endometrium, a high proportion of endometrial adenocarcinomas showed impaired HDAC-1 protein expression in the epithelial and stromal compartment. CONCLUSIONS: Class I HDACs and HATs are expressed in the human endometrium throughout the menstrual cycle, suggesting the cyclic endometrium as a potential target for HDAC inhibitors. We hypothesis that alterations of HDAC and/or HAT expression are potentially involved in impaired endometrial differentiation.

Expression of leucine-rich repeat-containing G-protein-coupled receptors in the human cyclic endometrium.

OBJECTIVE: To study the mRNA expression of the two leucine-rich repeat-containing G-protein-coupled receptors LGR-4 and LGR-5 and the mRNA and protein expression of LGR-7, the relaxin receptor, in the human cyclic endometrium. DESIGN: Retrospective study. SETTING: Department of Anatomy and Reproductive Biology, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany. METHOD(S): LGR-4, -5, and -7 mRNA expression was assessed by semiquantitative and real time reverse transcription-polymerase chain reaction in the endometrium of premenopausal women (n = 26) and cultured primary endometrial epithelial cells and fibroblasts (n = 3). Transcript size was determined by Northern blotting. LGR-7 protein expression was assessed by immunohistochemistry. RESULT(S): The mRNA of LGR-4, LGR-5, and LGR-7 was expressed constitutively throughout the menstrual cycle in the endometrium, and characterized by substantial differences in expression levels of individual women. LGR-7 immunostaining was detected in the epithelium of the functional layer throughout the cycle, with lowest staining in the midproliferative phase. Furthermore, individual stromal cells of the functional layer and the stroma of the basal layer showed LGR-7 immunostaining. CONCLUSION(S): Endometrial expression of the mRNA of orphan receptors LGR-4 and LGR-5 implies that the endometrium is potentially influenced by as yet unknown mediators, which are possibly involved in fertility control. Furthermore, we confirmed constitutive endometrial mRNA expression of LGR-7, the classical relaxin receptor, and demonstrated specific LGR-7 immunostaining of different endometrial cell types, which suggests a physiological role of relaxin in the human endometrium.