Christian August

First evidence supporting a potential role for the BMP/SMAD pathway in the progression of oestrogen receptor-positive breast cancer

Oestrogen receptor expression is generally a sign of better tumour differentiation and comparatively good clinical outcome in invasive breast cancer. However, oestrogen receptor‐positive, poorly differentiated carcinomas with a poor clinical outcome exist. The underlying genetic mechanisms and the genes involved remain obscure, even though chromosome 7p gains seem to be associated with these uncommon tumours. In this study, we compared two subsets of oestrogen receptor‐positive breast cancers, which differed in tumour grade, cytogenetic instability, and tumour proliferation, for their differential gene expression in order to identify proteins involved in the progression of oestrogen receptor‐positive breast cancers. We were able to show by means of subtractive suppression hybridization, real‐time reverse transcriptase PCR, and tissue microarray analysis that expression of the bone morphogenetic protein receptor IB (BMPR‐IB) is a major hallmark of the progression and dedifferentiation of breast cancer. Strong expression of BMPR‐IB was associated with high tumour grade, high tumour proliferation, cytogenetic instability, and a poor prognosis in oestrogen receptor‐positive carcinomas. Western blot analysis revealed that downstream signalling of this receptor is mainly mediated via phosphorylation of SMAD 1 in oestrogen receptor‐positive breast cancer. Even though BMPR‐IB was expressed in oestrogen receptor‐positive and ‐negative breast cancers, an impact on tumour grade, proliferation, and cytogenetic instability, as parameters of tumour progression, could only be demonstrated in oestrogen receptor‐positive carcinomas. This pro‐proliferative effect was complemented by significant anti‐apoptotic activity, indicated by XIAP and IAP‐2 expression in BMPR‐IB‐positive carcinomas. These results show that the BMP/SMAD pathway is activated in breast cancer and may contribute to breast cancer progression and dedifferentiation in oestrogen receptor‐positive breast cancer. The definition of this pathway characterizes a new potential target in the molecular treatment of invasive breast cancer. Copyright

CGH and CD 44/MIB-1 immunohistochemistry are helpful to distinguish metastasized from nonmetastasized sporadic pheochromocytomas

The natural course of pheochromocytomas (PCC) cannot be predicted for certain on the basis of primary histology, their malignant character can only be confirmed by the occurrence of metastases during follow-up. Based on the recently proposed PASS score for evaluation we examined 37 adrenal (36 sporadic and one familial) and six sporadic extra-adrenal paragangliomas (all designated as pheochromocytomas) with a ‘malignant histology’ to find additional predictive factors. Drawing upon the follow-up (18 months to 12 years, mean 5.8 years) metastasized (n=20) and nonmetastasized (n=23) courses could be distinguished. Metastasized PCC revealed significantly (P=0.03) more copy number changes on comparative genomic hybridization (CGH) (mean 8.3) than nonmetastasized tumors (mean: 4.3). The most frequent chromosomal alterations were losses on 1p (75.6%) and 3q (44%). Both were detected with identical frequency in metastasized and nonmetastasized PCC. A gain on 17q (P=0.025) was significantly predominant in malignant courses and suggests similarities in the genetic origin and progression of PCC and neuroblastomas. The proliferative activity (MIB-1 score) of metastasized PCC (n=20) was found to be significantly higher in metastasized tumors (mean 12.8% vs mean 3.5%). In contrast, the semiquantitatively scored membrane-bound staining of CD 44-S was stronger in tumors without metastases (mean 2.1 vs mean: 0.25) during the follow-up period (P<0.01). Although the results correspond to the established weight differences the tumor weight does not appear to be an independent prognostic factor. Our study suggests that CD 44-S and MIB-1 immunostaining as well as the CGH results might complement the PASS score in predicting a metastasized course of PCC. Regardless of tumor weight, tumors with a ‘malignant histology’ are highly prone to metastasize when more than 5% of MIB1-positive nuclei are present or CD44-S immunostaining is negative, or both. PCC with 10 or more copy number changes on CGH must be referred to as malignant tumors.

Reversal of metallothionein expression is different throughout the human myocardium after prolonged left-ventricular mechanical support

OBJECTIVES We examined the distribution of metallothionein (MT), a stress-inducible protein, and the cardiomyocyte diameter in human hearts after left-ventricular assist device (LVAD) support. BACKGROUND Remodeling in end-stage heart failure is characterized by myocyte hypertrophy and alterations of several inducible proteins. LVADs used as a bridge to cardiac transplantation unload the left ventricle and may lead to a reversal of the remodeling, but little is known about the pathophysiology of this process. METHODS The immunoreactivity for MT and the cardiomyocyte diameter was analyzed in left-ventricular tissue specimens of 17 patients with end-stage heart failure before and after LVAD support. RESULTS MT positive cells were mainly located sub-endocardially in vacuolized cardiomyocytes and in small vessels throughout the myocardium. During LVAD support, MT-positive myocytes decreased in the sub-endocardial (p < 0.008) and sub-epicardial region (p < 0.003), MT-positive vessels decreased similarly (p < 0.003). Cardiomyocyte diameter decreased significantly only in the sub-endocardium (p < 0.03). Hearts of patients supported longer than 88 days (= median) showed substantially lower MT reactivity at the time of LVAD explantation as compared to patients supported less than 88 days. CONCLUSION Our results suggest that unloading of the left ventricle during prolonged LVAD support leads to regression of cellular hypertrophy and a decrease of MT expression. The preferential reduction of MT-positive vacuolized cardiomyocytes in the sub-endocardium is comparable with the concept of greatest reduction of wall stress in this area of the myocardium and may be due to the improvement of myocardial blood flow and the energy balance.

Expression of β-catenin and p53 are prognostic factors in deep aggressive fibromatosis

Aims:  To determine the prognostic significance of β‐catenin in aggressive fibromatosis and to identify potential molecular markers for new targeted therapies.

Down-Regulation of Monocyte Apoptosis by Phagocytosis of Platelets: Involvement of a Caspase-9, Caspase-3, and Heat Shock Protein 70-Dependent Pathway

Monocytes interact and cross-talk with platelets in many settings including inflammation, hemostasis, or vascular disorders. During inflammatory diseases, there is a rapid targeting of monocytes and platelets to points of inflammation and endothelial injury, where they lie side-by-side. In this in vitro study, we investigated different interactions between monocytes and platelets and elucidated whether platelets might affect monocyte apoptosis. Freshly isolated human monocytes were rendered apoptotic by serum deprivation or CD95 ligation and cocultured with platelets. Monocyte apoptosis was determined by flow cytometry, TUNEL staining, DNA electrophoresis, and transmission electron microscopy imaging. We could show that monocyte apoptosis was highly suppressed when platelets were added to the cultures. Transmission electron microscopy depicted that monocytes completely ingested thrombocytes by phagocytosis. Blocking thrombocyte uptake by the phagocytosis inhibitor cytochalasin D abrogated the enhanced monocyte survival and led to high apoptosis levels. Monocyte survival was paralleled by down-regulation of caspase-9 and -3 and up-regulation of heat shock protein 70 during uptake of platelets. Platelet supernatants and contents of platelet granules were ineffective in altering monocyte senescence. Also, ingestion of latex beads or zymosan by monocytes was ineffective to mimic platelet-dependent rescue from apoptosis. In conclusion, this study shows that platelets can suppress apoptosis of monocytes by a specific phagocytosis-dependent process with further consequences for atherosclerotic or inflammatory conditions.

Early multiple organ failure after recurrent endotoxemia in the presence of vasoconstrictor-masked hypovolemia.

ObjectiveCritically ill patients who develop multiple organ failure during systemic inflammatory states are often predisposed to hypovolemia and vasoconstrictor therapy. Although numerous investigations have evaluated the sequelae of systemic inflammation, no data are available on the contribution of chronic vasoconstrictor-masked hypovolemia to organ dysfunction and morphology. DesignProspective, randomized laboratory investigation. SettingUniversity research laboratory. SubjectsEighteen adult chronically instrumented sheep. InterventionsThe animals were randomly assigned to one of three groups. In the norfenefrine-masked hypovolemia plus endotoxemia (NMH+ENDO) group, mean arterial pressures of 80 mm Hg were maintained by using the &agr;1-adrenergic catecholamine norfenefrine for 52 hrs during hypovolemia. Hypovolemia was induced by hemorrhage (about 23 mL·kg−1) until mean arterial pressures reached 40 mm Hg. Endotoxin (0.5 &mgr;g·kg−1) was then injected after 4, 16, 28, and 40 hrs. The NMH group received norfenefrine-masked hypovolemia but no endotoxin. In the ENDO group, recurrent endotoxemia was induced during normovolemia. Measurements and Main ResultsDespite profound differences in fluid management, cardiovascular filling pressures were not statistically different between groups. Endotoxemia induced norfenefrine-refractory shock (p < .05 vs. the other groups) and contributed to renal dysfunction only during vasoconstrictor-masked hypovolemia. Norfenefrine-masked hypovolemia caused disseminated cardiac cell necrosis independent of endotoxemia (p < .05 vs. ENDO). ConclusionsHypovolemia can be masked when volume status is monitored by filling pressures. In this new model of endotoxemia-associated multiple organ failure, chronic vasoconstrictor-masked hypovolemia turned systemic inflammation into a life-threatening condition with renal and cardiovascular failure. Cardiomyocyte necroses were caused by vasoconstrictor-masked hypovolemia but were unrelated to cardiovascular failure.

Differential regulation of glomerular gelatinase B (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in obese Zucker rats

Summary The obese Zucker rat represents a model of obesity combined with insulin resistance and hyperlipidaemia, which over a period of several months develops spontaneous glomerulosclerosis. The present study addressed the question as to whether glomerular sclerosis was associated with alterations in the degradation of matrix components. In the early phase (up to 6 months) glomeruli from obese rats displayed increased total collagen content (+ 64 %) and decreased gelatinolytic activity (− 34 %) as compared to lean control animals. This decline in glomerular gelatinolytic activity was due to a reduction in gelatinase B [matrix metalloproteinase (MMP)-9]. Glomerular MMP-9 mRNA was reduced 4.6 ± 0.6-fold (n = 3; p < 0.05), MMP-9 protein was not detectable by Western blotting and MMP-9 activity was considerably suppressed in gelatin zymograms. MMP-2, in terms of mRNA expression and activity, was unchanged. Tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression, TIMP-1 protein (immunohistochemistry) and TIMP-1 activity (reverse zymography) were enhanced in glomeruli from obese rats, while TIMP-2 mRNA remained unchanged. Moreover, mRNA for the α1 IV collagen chain was 2.1 ± 0.8-fold higher in glomeruli isolated from obese animals (n = 3; p < 0.05). These findings indicate that matrix expansion in glomeruli from obese Zucker rats is due to both enhanced synthesis of matrix components as well as reduced degradation by matrix metalloproteinases. Apparently the latter effect is based on a reduction in MMP-9 and up-regulation of its inhibitor TIMP-1. [Diabetologia (1997) 40: 1035–1043]

Reduction of hypoxia‐inducible heme oxygenase‐1 in the myocardium after left ventricular mechanical support

Left ventricular assist devices (LVAD) may improve cardiac function. The pathogenesis of this phenomenon, called ‘reverse remodelling’, is not completely elucidated. To examine the hypothesis that LVAD support eliminates tissue stress by reducing local hypoxia, the distribution of heme oxygenase‐1 (HO‐1), a stress protein inducible by hypoxia, was examined in vivo and in vitro. The immunoreactivity for HO‐1 was semi‐quantitatively analysed in left ventricular tissue of 23patients (14 dilated cardiomyopathy (DCM), six ischaemic heart disease (IHD), three myocarditis/congenital heart disease) with end‐stage heart failure before and after LVAD support, while two unused donor hearts served as controls. Control hearts stained almost negative for HO‐1, while failing hearts showed immunoreactivity mainly in cardiomyocytes, but also in endothelial cells, some smooth muscle cells and fibroblasts. Hearts with IHD showed significantly higher HO‐1 immunoreactivity than hearts with DCM or myocarditis/congenital heart disease. After LVAD support, the HO‐1 content decreased significantly in the DCM and IHD group and was significantly higher in the subendocardium than in the subepicardium. In vitro, under hypoxic conditions, neonatal rat cardiomyocytes showed an increase of HO‐1 protein content up to sixfold above the normal level, which returned to normal values after normoxic cultivation. Mechanical support reduces the HO‐1 content of the failing heart and HO‐1 is inducible in vitro under hypoxia and is reversible under normoxia. This supports the concept that restoration of cardiac normoxia by mechanical unloading, particularly in the subendocardium, may be in part responsible for the phenomenon of ‘reverse remodelling’. Copyright

A short-term in vivo model for giant cell tumor of bone

BackgroundBecause of the lack of suitable in vivo models of giant cell tumor of bone (GCT), little is known about its underlying fundamental pro-tumoral events, such as tumor growth, invasion, angiogenesis and metastasis. There is no existing cell line that contains all the cell and tissue tumor components of GCT and thus in vitro testing of anti-tumor agents on GCT is not possible. In this study we have characterized a new method of growing a GCT tumor on a chick chorio-allantoic membrane (CAM) for this purpose.MethodsFresh tumor tissue was obtained from 10 patients and homogenized. The suspension was grafted onto the CAM at day 10 of development. The growth process was monitored by daily observation and photo documentation using in vivo biomicroscopy. After 6 days, samples were fixed and further analyzed using standard histology (hematoxylin and eosin stains), Ki67 staining and fluorescence in situ hybridization (FISH).ResultsThe suspension of all 10 patients formed solid tumors when grafted on the CAM. In vivo microscopy and standard histology revealed a rich vascularization of the tumors. The tumors were composed of the typical components of GCT, including (CD51+/CD68+) multinucleated giant cells whichwere generally less numerous and contained fewer nuclei than in the original tumors. Ki67 staining revealed a very low proliferation rate. The FISH demonstrated that the tumors were composed of human cells interspersed with chick-derived capillaries.ConclusionsA reliable protocol for grafting of human GCT onto the chick chorio-allantoic membrane is established. This is the first in vivo model for giant cell tumors of bone which opens new perspectives to study this disease and to test new therapeutical agents.

Impact of renal transplantation on small vessel reactivity.

Background. The function of large arteries is altered after renal transplantation. Whether transplantation also induces agonist-dependent functional changes in small arterial renal and extrarenal vessels has not yet been studied. Methods. Chronic rejection was induced by grafting Lewis rats with kidneys from Fischer rats (FL). Rats that underwent transplantation were bilaterally nephrectomized. Rats that underwent syngeneic transplantation, uninephrectomized rats, uninephrectomized rats with denervated kidneys or with kidneys made ischemic, and native rats served as controls. All animals were treated with cyclosporine for 10 days. Eighteen weeks after surgery, the reactivity of small arteries (220–270 &mgr;m) was tested by myography. Results. Weight gain, glomerular filtration rate, and arterial pressure were similar in all groups, whereas proteinuria was elevated in FL. Only kidneys from FL showed glomerular lesions, tubular atrophy, and vasculopathy. Responsiveness of coronary, mesenteric, and femoral resistance vessels to both constrictor and dilator agonists was similar in transplanted and nontransplanted animals. Resistance vessels obtained from both allogeneically and syngeneically transplanted kidneys were more sensitive to norepinephrine, phenylephrine, angiotensin II, and vasopressin than renal vessels from weight-matched controls. Vasodilation in response to acetylcholine and sodium nitroprusside was mitigated in transplanted versus nontransplanted kidneys. Conclusions. In rat renal transplantation, renal resistance vessel responsiveness to constrictor or dilator stimuli is altered. Extrarenal small vessel function is not affected. The changes in function of renal resistance vessels are not explained by reduction of nephron mass, denervation, ischemia, or chronic rejection.