BMP9 and BMP10 Act Directly on Vascular Smooth Muscle Cells for Generation and Maintenance of the Contractile State.

BACKGROUND: Vascular smooth muscle cells (VSMCs) show a remarkable phenotypic plasticity, allowing acquisition of contractile or synthetic states, but critical information is missing about the physiologic signals, promoting formation, and maintenance of contractile VSMCs in vivo. BMP9 and BMP10 (bone morphogenetic protein) are known to regulate endothelial quiescence after secretion from the liver and right atrium, whereas a direct role in the regulation of VSMCs was not investigated. We studied the role of BMP9 and BMP10 for controlling formation of contractile VSMCs. METHODS: We generated several cell type-specific loss- and gain-of-function transgenic mouse models to investigate the physiologic role of BMP9, BMP10, ALK1 (activin receptor-like kinase 1), and SMAD7 in vivo. Morphometric assessments, expression analysis, blood pressure measurements, and single molecule fluorescence in situ hybridization were performed together with analysis of isolated pulmonary VSMCs to unravel phenotypic and transcriptomic changes in response to absence or presence of BMP9 and BMP10. RESULTS: Concomitant genetic inactivation of Bmp9 in the germ line and Bmp10 in the right atrium led to dramatic changes in vascular tone and diminution of the VSMC layer with attenuated contractility and decreased systemic as well as right ventricular systolic pressure. On the contrary, overexpression of Bmp10 in endothelial cells of adult mice dramatically enhanced formation of contractile VSMCs and increased systemic blood pressure as well as right ventricular systolic pressure. Likewise, BMP9/10 treatment induced an ALK1-dependent phenotypic switch from synthetic to contractile in pulmonary VSMCs. Smooth muscle cell-specific overexpression of Smad7 completely suppressed differentiation and proliferation of VSMCs and reiterated defects observed in adult Bmp9/10 double mutants. Deletion of Alk1 in VSMCs recapitulated the Bmp9/10 phenotype in pulmonary but not in aortic and coronary arteries. Bulk expression analysis and single molecule RNA-fluorescence in situ hybridization uncovered vessel bed-specific, heterogeneous expression of BMP type 1 receptors, explaining phenotypic differences in different Alk1 mutant vessel beds. CONCLUSIONS: Our study demonstrates that BMP9 and BMP10 act directly on VSMCs for induction and maintenance of their contractile state. The effects of BMP9/10 in VSMCs are mediated by different combinations of BMP type 1 receptors in a vessel bed-specific manner, offering new opportunities to manipulate blood pressure in the pulmonary circulation.

Inhibition of Notch2 by Numb/Numblike controls myocardial compaction in the heart.

AIMS: The ventricular wall of the heart is composed of trabeculated and compact layers, which are separated by yet unknown processes during embryonic development. Here, we wanted to explore the role of Notch2 and Numb/Numblike for myocardial trabeculation and compaction. METHODS AND RESULTS: We found that Notch2 activity is specifically down-regulated in the compact layer during cardiac development in the mouse. The biological role of Notch2 down-regulation was investigated by the expression of constitutively active Notch2 in the myocardium of transgenic mice, resulting in hypertrabeculation, reduced compaction, and ventricular septum defects. To disclose the mechanism that inhibited Notch2 activity during the formation of myocardial layers, we analysed potential suppressors of Notch signalling. We unveiled that concomitant but not separate ablation of Numb and Numblike in the developing heart leads to increased Notch2 activity along with hypertrabeculation, reduced compaction, and ventricular septum defects, phenocopying effects gained by overexpression of constitutively active Notch2. Expression profiling revealed a strong up-regulation of Bmp10 in Numb/Numblike mutant hearts, which might also interfere with trabeculation and compaction. CONCLUSION: This study identified potential novel roles of Numb/Numblike in regulating trabeculation and compaction by inhibiting Notch2 and Bmp10 signalling.

Cardiomyocyte-specific IκB kinase (IKK)/NF-κB activation induces reversible inflammatory cardiomyopathy and heart failure.

Inflammation is a major factor in heart disease. IκB kinase (IKK) and its downstream target NF-κB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-κB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2. In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b(+) cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. Upon transgene inactivation, the disease was reversible even at an advanced stage. IKK-induced cardiomyopathy was dependent on NF-κB activation, as in vivo expression of IκBα superrepressor, an inhibitor of NF-κB, prevented the development of disease. Gene expression and proteomic analyses revealed enhanced expression of inflammatory cytokines, and an IFN type I signature with activation of the IFN-stimulated gene 15 (ISG15) pathway. In that respect, IKK-induced cardiomyopathy resembled Coxsackievirus-induced myocarditis, during which the NF-κB and ISG15 pathways were also activated. Vice versa, in cardiomyocytes lacking the regulatory subunit of IKK (IKKγ/NEMO), the induction of ISG15 was attenuated. We conclude that IKK/NF-κB activation in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure by inducing an excessive inflammatory response and myocyte atrophy.

The Janus face of OSM-mediated cardiomyocyte dedifferentiation during cardiac repair and disease.

Dedifferentiation is a common phenomenon among plants but has only been found rarely in vertebrates where it is mostly associated with regenerative responses such as formation of blastemae in amphibians to initiate replacement of lost body parts. Relatively little attention has been paid to dedifferentiation processes in mammals although a decline of differentiated functions and acquisition of immature, "embryonic" properties is seen in various disease processes. Dedifferentiation of parenchymal cells in mammals might serve multiple purposes including (1) facilitation of tissue regeneration by generation of progenitor-like cells and (2) protection of cells from hypoxia by reduction of ATP consumption due to changes in energy metabolism and/or inactivation of energy-intensive "specialized" functions. We recently found that an inflammatory cytokine of the interleukin 6 family, oncostatin M (OSM), initiates dedifferentiation of cardiomyocytes both in vitro and in vivo. Interestingly, activation of the OSM signaling pathway protects the heart from acute myocardial ischemia but has a negative impact when continuously activated thereby promoting dilative cardiomyopathy. The strong presence of the OSM receptor on cardiomyocytes and the unique features of the OSM signaling circuit suggest a major role of OSM for cardiac protection and repair. We propose that continuous activation or malfunctions of the cellular dedifferentiation machinery might contribute to different disease conditions.

Obesity-induced overexpression of miRNA-143 inhibits insulin-stimulated AKT activation and impairs glucose metabolism.

The contribution of altered post-transcriptional gene silencing to the development of insulin resistance and type 2 diabetes mellitus so far remains elusive. Here, we demonstrate that expression of microRNA (miR)-143 and 145 is upregulated in the liver of genetic and dietary mouse models of obesity. Induced transgenic overexpression of miR-143, but not miR-145, impairs insulin-stimulated AKT activation and glucose homeostasis. Conversely, mice deficient for the miR-143-145 cluster are protected from the development of obesity-associated insulin resistance. Quantitative-mass-spectrometry-based analysis of hepatic protein expression in miR-143-overexpressing mice revealed miR-143-dependent downregulation of oxysterol-binding-protein-related protein (ORP) 8. Reduced ORP8 expression in cultured liver cells impairs the ability of insulin to induce AKT activation, revealing an ORP8-dependent mechanism of AKT regulation. Our experiments provide direct evidence that dysregulated post-transcriptional gene silencing contributes to the development of obesity-induced insulin resistance, and characterize the miR-143-ORP8 pathway as a potential target for the treatment of obesity-associated diabetes.

Cardiac deletion of Smyd2 is dispensable for mouse heart development.

Chromatin modifying enzymes play a critical role in cardiac differentiation. Previously, it has been shown that the targeted deletion of the histone methyltransferase, Smyd1, the founding member of the SET and MYND domain containing (Smyd) family, interferes with cardiomyocyte maturation and proper formation of the right heart ventricle. The highly related paralogue, Smyd2 is a histone 3 lysine 4- and lysine 36-specific methyltransferase expressed in heart and brain. Here, we report that Smyd2 is differentially expressed during cardiac development with highest expression in the neonatal heart. To elucidate the functional role of Smyd2 in the heart, we generated conditional knockout (cKO) mice harboring a cardiomyocyte-specific deletion of Smyd2 and performed histological, functional and molecular analyses. Unexpectedly, cardiac deletion of Smyd2 was dispensable for proper morphological and functional development of the murine heart and had no effect on global histone 3 lysine 4 or 36 methylation. However, we provide evidence for a potential role of Smyd2 in the transcriptional regulation of genes associated with translation and reveal that Smyd2, similar to Smyd3, interacts with RNA Polymerase II as well as to the RNA helicase, HELZ.

Expression profiling of cardiac genes in Tako-Tsubo cardiomyopathy: insight into a new cardiac entity.

Tako-Tsubo cardiomyopathy (TTC) is characterized by a transient contractile dysfunction, but its specific pathomechanism remains unknown. Thus, we performed a systematic expression profiling of genes by microarray analysis in the acute phase and after functional recovery. We studied 3 female patients presenting with TTC. Complementary RNA was isolated from left ventricular biopsies taken in the acute phase (group A) and after functional recovery (group B). It was profiled for gene expression using cDNA microarrays. Functionally related genes were determined with the Gene Set Enrichment Analysis (GSEA) bioinformatic tool. Validation of selected genes was performed by means of real-time PCR and immunohistochemistry. In group A, different functional gene sets, such as Nrf2-induced genes, triggered by oxidative stress, and protein biosynthesis were significantly overrepresented among the upregulated targets. Increased transcription of GPX1, CAT, RPS6, and eIF4E was confirmed by RT-PCR. The targets of the Akt/PKB signaling showed significant upregulation in both groups. Immunohistochemistry showed that the downstream targets NF-kappaB and BcL-X(L) are upregulated and activated. Gene sets involved in energy metabolism (oxidative phosphorylation, mitochondrial genes) showed no differences in group A but were overexpressed in group B. This study demonstrated a significant contribution of oxidative stress to the pathomechanism of TTC; it is possibly triggered by excess catecholamine. Increased protein biosynthesis and an activated cell survival cascade can be interpreted as potential compensatory mechanisms. After functional recovery, processes involved in energy metabolism play a pivotal role, thereby potentially contributing to the normalization of contractile function.

Application of a new segmentation tool based on interactive simplex meshes to cardiac images and pulmonary MRI data.

RATIONALE AND OBJECTIVES: Medical image segmentation is still very time consuming and is therefore seldom integrated into clinical routine. Various three-dimensional (3D) segmentation approaches could facilitate the work, but they are rarely used in clinical setups because of complex initialization and parametrization of such models. MATERIALS AND METHODS: We developed a new semiautomatic 3D-segmentation tool based on deformable simplex meshes. The user can define attracting points in the original image data. The new deformation algorithm guarantees that the surface model will pass through these interactively set points. The user can directly influence the evolution of the deformable model and gets direct feedback during the segmentation process. RESULTS: The segmentation tool was evaluated for cardiac image data and magnetic resonance imaging lung images. Comparison with manual segmentation showed high accuracy. Time needed for delineation of the various structures could be reduced in some cases. The model was not sensitive to noise in the input data and model initialization. CONCLUSIONS: The tool is suitable for fast interactive segmentation of any kind of 3D or 3D time-resolved medical image data. It enables the clinician to influence a complex 3D-segmentation algorithm and makes this algorithm controllable. The better the quality of the data, the less interaction is required. The tool still works when the processed images have low quality.

The medical imaging interaction toolkit.

Thoroughly designed, open-source toolkits emerge to boost progress in medical imaging. The Insight Toolkit (ITK) provides this for the algorithmic scope of medical imaging, especially for segmentation and registration. But medical imaging algorithms have to be clinically applied to be useful, which additionally requires visualization and interaction. The Visualization Toolkit (VTK) has powerful visualization capabilities, but only low-level support for interaction. In this paper, we present the Medical Imaging Interaction Toolkit (MITK). The goal of MITK is to significantly reduce the effort required to construct specifically tailored, interactive applications for medical image analysis. MITK allows an easy combination of algorithms developed by ITK with visualizations created by VTK and extends these two toolkits with those features, which are outside the scope of both. MITK adds support for complex interactions with multiple states as well as undo-capabilities, a very important prerequisite for convenient user interfaces. Furthermore, MITK facilitates the realization of multiple, different views of the same data (as a multiplanar reconstruction and a 3D rendering) and supports the visualization of 3D+t data, whereas VTK is only designed to create one kind of view of 2D or 3D data. MITK reuses virtually everything from ITK and VTK. Thus, it is not at all a competitor to ITK or VTK, but an extension, which eases the combination of both and adds the features required for interactive, convenient to use medical imaging software. MITK is an open-source project (www.mitk.org).

Programmable laser frequency stabilization at 1523 nm by use of persistent spectral hole burning.

Diode laser frequency stability of 2 kHz to 680 Hz over 20 ms to 500 s has been demonstrated at 1523 nm in the technologically important communication band by use of persistent spectral holes in the inhomogeneously broadened 4I15/2 --> 4I13/2 optical absorption of Er3+:D-:CaF2. Laser frequency stabilization was realized without vibrational or acoustical isolation of either the laser or spectral hole frequency reference, providing the means for implementing a versatile, compact, stable source.

How many lymph nodes are needed for an accurate pN classification in esophageal cancer? Evidence for a new threshold value.

BACKGROUND/AIMS: The UICC recommends a number of at least six lymph nodes to be examined in the surgical therapy of esophageal cancer for a reliable pN classification. The aim of this study was to evaluate this threshold by means of the data from our patients. METHODOLOGY: Following curative resection (R0) of esophageal cancer the numbers of examined tumor-free and tumor-involved lymph nodes were compared. Different statistical models of logistic regression were fitted to the data and checked for plausibility (Hosmer Lemeshow test). The sensitivity of a correct pN classification was then calculated and correlated to the total number of examined lymph nodes. RESULTS: A maximum increase of the sensitivity in classifying pN occurred from 0 to 6 examined lymph nodes. Nevertheless an additional improvement of sensitivity was continuously shown up to 100 examined nodes. An over 90% sensitivity of a correct lymph node classification was reached when more than twelve nodes were examined. Thus the results demonstrate in the case of esophageal cancer, that the suggestion by the UICC to examine at least 6 nodes for defining pN appears too low and may not represent the clinical situation. A ninety percent confidence level of a correct lymph node classification can be expected above 12 examined nodes similarly to the current recommended threshold in colorectal carcinoma. CONCLUSIONS: We suggest a new threshold for the number of examined lymph nodes of at least 12 instead of 6 nodes for accurately defining the pN category in esophageal cancer.