During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1 1 precursors. Embryos deficient for Nkx2-5 in the Isl1 1 lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.

Two types of distinct cardiac progenitor cell populations can be identified during early heart development: the first heart field (FHF) and second heart field (SHF) lineage that later form the mature heart. They can be characterized by differential expression of transcription and signaling factors. These regulatory factors influence each other forming a gene regulatory network. Here, we present a core gene regulatory network for early cardiac development based on published temporal and spatial expression data of genes and their interactions. This gene regulatory network was implemented in a Boolean computational model. Simulations reveal stable states within the network model, which correspond to the regulatory states of the FHF and the SHF lineages. Furthermore, we are able to reproduce the expected temporal expression patterns of early cardiac factors mimicking developmental progression. Additionally, simulations of knock-down experiments within our model resemble published phenotypes of mutant mice. Consequently, this gene regulatory network retraces the early steps and requirements of cardiogenic mesoderm determination in a way appropriate to enhance the understanding of heart development.

Nintedanib inhibits proliferation of pulmonary MVECs from controls, but not from PAH patients. While in rats with experimental PH nintedanib has no effects on the pulmonary vascular pathology, it has favorable effects on right ventricular remodeling.

The anti-inflammatory and immunomodulatory abilities of oral selective phosphodiesterase 4 (PDE4) inhibitors enabled the approval of roflumilast and apremilast for use in chronic obstructive pulmonary disease and psoriasis/psoriatic arthritis, respectively. However, the antifibrotic potential of PDE4 inhibitors has not yet been explored clinically. BI 1015550 is a novel PDE4 inhibitor showing a preferential enzymatic inhibition of PDE4B. In vitro, BI 1015550 inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and phytohemagglutinin-induced interleukin-2 synthesis in human peripheral blood mononuclear cells, as well as LPS-induced TNF-α synthesis in human and rat whole blood. In vivo, oral BI 1015550 shows potent anti-inflammatory activity in mice by inhibiting LPS-induced TNF-α synthesis ex vivo and in Suncus murinus by inhibiting neutrophil influx into bronchoalveolar lavage fluid stimulated by nebulized LPS. In Suncus murinus, PDE4 inhibitors induce emesis, a well-known gastrointestinal side effect limiting the use of PDE4 inhibitors in humans, and the therapeutic ratio of BI 1015550 appeared to be substantially improved compared with roflumilast. Oral BI 1015550 was also tested in two well-known mouse models of lung fibrosis (induced by either bleomycin or silica) under therapeutic conditions, and appeared to be effective by modulating various model-specific parameters. To better understand the antifibrotic potential of BI 1015550 in vivo, its direct effect on human fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) was investigated in vitro. BI 1015550 inhibited transforming growth factor-β-stimulated myofibroblast transformation and the mRNA expression of various extracellular matrix proteins, as well as basic fibroblast growth factor plus interleukin-1β-induced cell proliferation. Nintedanib overall was unremarkable in these assays, but interestingly, the inhibition of proliferation was synergistic when it was combined with BI 1015550, leading to a roughly 10-fold shift of the concentration–response curve to the left. In summary, the unique preferential inhibition of PDE4B by BI 1015550 and its anticipated improved tolerability in humans, plus its anti-inflammatory and antifibrotic potential, suggest BI 1015550 to be a promising oral clinical candidate for the treatment of IPF and other fibro-proliferative diseases.

The T-box transcription factor Tbx5 is involved in several developmental processes including cardiogenesis. Early steps of cardiac development are characterised by the formation of two cardiogenic lineages, the first (FHF) and the second heart field (SHF) lineage, which arise from a common cardiac progenitor cell population. To further investigate the function of Tbx5 during cardiogenesis, we generated a murine embryonic stem cell line constitutively overexpressing Tbx5. Differentiation of these cells is characterised by an earlier and increased appearance of contracting cardiomyocytes that beat with a higher frequency than control cells. In semi-quantitative and quantitative RT-PCR analyses, we observed an up-regulation of cardiac marker genes such as Troponin T, endogenous Tbx5, and Nkx2.5 and a down-regulation of others like BMP4 and Hand2. Similar data were gained in Xenopus laevis arguing for a conserved function of Tbx5. Furthermore, markers of the conduction system and atrial cardiomyocytes were increased.

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Until recently it was believed that the T cell response of atopic dermatitis patients challenged with inhalant allergens originates almost exclusively and specifically from Th2 cells capable of secreting an abundance of interleukin (IL)-4 while producing no interferon (IFN)-gamma. To reevaluate this concept in a large cohort of atopic dermatitis patients we established 177 CD4+ T cell clones (45 of which showed specificity for house dust mite antigen) from the peripheral blood (n = 76), naturally occurring skin lesions (n = 40), and allergen-exposed skin (n = 61) of different patients. These clones were examined for their capacity to secrete IL-4 and IFN-gamma upon mitogenic stimulation. Moreover, 20 of these T cell clones were investigated for the synthesis of transcripts for IL-5, another Th cytokine. Our results indicate that the majority (52-100%) of allergen-specific T cells in both skin and blood of atopic individuals failed to exhibit a restricted cytokine secretion pattern and thus were classified as Th0 cells. House dust mite antigen specific T cells displaying a restricted secretion pattern (n = 16) were either of the Th1 or the Th2 type. Specific Th2 cells, however, were found almost exclusively in allergen patch test reactions, indicating that the Th2 differentiation pathway is seen preferentially in allergen-exposed skin. The cytokine secretion profile of T cell clones obtained from naturally occurring skin lesions showed similarity to those of patch test lesion, suggesting that the patch test represents a useful model to investigate the pathogenesis of atopic dermatitis.

How cancer cells become resistant to chemotherapy is not completely understood, but it is believed that resistance is usually associated with overexpression of drug resistance genes. Drug resistance mediated by the MDR‐1 gene is the first well characterized form of drug resistance in human cancer. MDR‐1 encodes a phosphoglycoprotein, P‐GP, that serves as an energy‐dependent drug efflux pump, reducing intracellular drug accumulation and thereby cytotoxicity. We have used ribozymes to reverse the multiple drug resistance phenotype. A hammerhead ribozyme recognizing the GUC sequence at position ‐6 to ‐4 close to the translation start site of the 4.5 kb MDR‐1 mRNA was prepared by in vitro transcription (MDR‐1‐RZiv) or chemical synthesis (MDR‐1‐RZs). Both MDR‐1‐RZiv and MDR‐1‐RZs specifically cleaved the MDR‐1 mRNA into two parts of the expected size under physiological conditions in an extracellular system with MDR‐1‐RZiv being more effective. Site‐specific cleavage was dependent on time, temperature and [MgCl2]. To examine the in vivo potential of MDR‐1‐RZ, MDR‐1‐RZiv and MDR‐1‐RZs were transfected into a human pleural mesothelioma cell line and into one adriamycin‐resistant and one vindesine‐resistant subline thereof by liposome‐mediated transfer. Incorporation of ribozymes resulted in significantly reduced expression of the MDR‐1 gene, with MDR‐1‐RZs being more potent than MDR‐1‐RZiv in vitro. MDR‐1‐RZ reduces P‐GP overexpression at the protein level. Liposome‐mediated transfer of MDR‐1‐RZiv or MDR‐1‐RZs reversed the multiple drug resistance phenotype and restored sensitivity towards chemotherapeutic drugs.