Insulin-Like Growth Factor 1 Attenuates the Pro-Inflammatory Phenotype of Neutrophils in Myocardial Infarction.

Acute myocardial infarction (MI) induces an extensive sterile inflammation, which is dominated in the early phase by invading neutrophils and monocytes/macrophages. The inflammatory response after MI critically affects infarct healing and cardiac remodeling. Therefore, modulation of cardiac inflammation may improve outcome post MI. Insulin-like growth factor 1 (IGF1) treatment reduces infarct size and improves cardiac function after MI via IGF1 receptor mediated signaling in myeloid cells. Our study aimed to investigate the effect of IGF1 on neutrophil phenotype both in vitro and in vivo after MI. We show that IGF1 induces an anti-inflammatory phenotype in bone marrow derived neutrophils. On the molecular and functional level IGF1 treated neutrophils were indistinguishable from those induced by IL4. Surprisingly, insulin, even though it is highly similar to IGF1 did not create anti-inflammatory neutrophils. Notably, the IGF1 effect was independent of the canonical Ras/Raf/ERK or PI3K/AKT pathway, but depended on activation of the JAK2/STAT6 pathway, which was not activated by insulin treatment. Single cell sequencing analysis 3 days after MI also showed that 3 day IGF1 treatment caused a downregulation of pro-inflammatory genes and upstream regulators in most neutrophil and many macrophage cell clusters whereas anti-inflammatory genes and upstream regulators were upregulated. Thus, IGF1 acts like an anti-inflammatory cytokine on myeloid cells in vitro and attenuates the pro-inflammatory phenotype of neutrophils and macrophages in vivo after MI. IGF1 treatment might therefore represent an effective immune modulatory therapy to improve the outcome after MI.

4-hydroxytamoxifen does not deteriorate cardiac function in cardiomyocyte-specific MerCreMer transgenic mice.

Conditional, cell-type-specific transgenic mouse lines are of high value in cardiovascular research. A standard tool for cardiomyocyte-restricted DNA editing is the αMHC-MerCreMer/loxP system. However, there is an ongoing debate on the occurrence of cardiac side effects caused by unspecific Cre activity or related to tamoxifen/oil overload. Here, we investigated potential adverse effects of DNA editing by the αMHC-MerCreMer/loxP system in combination with a low-dose treatment protocol with the tamoxifen metabolite 4-hydroxytamoxifen (OH-Txf). αMHC-MerCreMer mice received intraperitoneally OH-Txf (20 mg/kg) for 5 or 10 days. These treatment protocols were highly efficient to induce DNA editing in adult mouse hearts. Multi-parametric magnetic resonance imaging revealed neither transient nor permanent effects on cardiac function during or up to 19 days after 5 day OH-Txf treatment. Furthermore, OH-Txf did not affect cardiac phosphocreatine/ATP ratios assessed by in vivo 31P MR spectroscopy, indicating no Cre-mediated side effects on cardiac energy status. No MRI-based indication for the development of cardiac fibrosis was found as mean T1 relaxation time was unchanged. Histological analysis of myocardial collagen III content after OH-Txf confirmed this result. Last, mean T2 relaxation time was not altered after Txf treatment suggesting no pronounced cardiac lipid accumulation or tissue oedema. In additional experiments, cardiac function was assessed for up to 42 days to investigate potential delayed side effects of OH-Txf treatment. Neither 5- nor 10-day treatment resulted in a depression of cardiac function. Efficient cardiomyocyte-restricted DNA editing that is free of unwanted side effects on cardiac function, energetics or fibrosis can be achieved in adult mice when the αMHC-MerCreMer/loxP system is activated by the tamoxifen metabolite OH-Txf.

IGF1 Treatment Improves Cardiac Remodeling after Infarction by Targeting Myeloid Cells.

Insulin-like growth factor 1 (IGF1) is an anabolic hormone that controls the growth and metabolism of many cell types. However, IGF1 also mediates cardio-protective effects after acute myocardial infarction (AMI), but the underlying mechanisms and cellular targets are not fully understood. Here we demonstrate that short-term IGF1 treatment for 3 days after AMI improved cardiac function after 1 and 4 weeks. Regional wall motion was improved in ischemic segments, scar size was reduced, and capillary density increased in the infarcted area and the border zone. Unexpectedly, inducible inactivation of the IGF1 receptor (IGF1R) in cardiomyocytes did not attenuate the protective effect of IGF1. Sequential cardiac transcriptomic analysis indicated an altered myeloid cell response in the acute phase after AMI, and, notably, myeloid-cell Igf1r-/- mice lost the protective IGF1 function after I/R. In addition, IGF1 induced an M2-like anti-inflammatory phenotype in bone marrow-derived macrophages and enhanced the number of anti-inflammatory macrophages in heart tissue on day 3 after AMI in vivo. In summary, modulation of the acute inflammatory phase after AMI by IGF1 represents an effective mechanism to preserve cardiac function after I/R.

Thrombin-induced ATP release from human umbilical vein endothelial cells.

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 µM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.

Horizontal gene transfer from human endothelial cells to rat cardiomyocytes after intracoronary transplantation.

AIMS: Recent studies suggested that human umbilical vein endothelial cells (HUVECs) transdifferentiate into cardiomyocytes and smooth muscle cells in vitro. To test the functional relevance of this observation, we examined the transdifferentiation potential of HUVECs in vivo after intracoronary cell application in Wistar rats. METHODS AND RESULTS: SPECT measurements (single photon emission computed tomography) revealed that 18% of (111)In-labelled HUVECs infused by intracoronary delivery stably transplanted to the rat heart. For long-term tracking, HUVECs-expressing enhanced green fluorescent protein (EGFP) were infused. Two days following transplantation, HUVECs were positive for caspase-3. Within 3 days, EGFP was associated with individual cardiomyocytes. No labelling of endothelial and smooth muscle cells was observed. The total number of EGFP-labelled cardiomyocytes accounted for 58% of all initially trapped cells. These EGFP positive cells stained negatively for human mitochondrial proteins, but were positive for rat monocarboxylate transporter-1 protein (MCT-1). Furthermore, EGFP-mRNA was detected in these cells by single-cell RT-PCR (reverse transcription followed by polymerase chain reaction). After 21 days, EGFP positive cells were no longer observed. To investigate the underlying mechanism, we generated in vitro apoptotic bodies from EGFP-labelled HUVECs and found them to contain the genetic information for EGFP. Co-incubation of apoptotic bodies with neonatal rat cardiomyocytes caused cardiomyocytes to express EGFP. CONCLUSION: When transplanted into the rat heart by efficient intracoronary delivery, EGFP-expressing HUVECs cause the exclusive but transient labelling of cardiomyocytes. Our in vivo findings suggest that it is not cell fusion and/or transdifferentiation that occurs under these conditions but rather a horizontal gene transfer of the EGFP marker via apoptotic bodies from endothelial cells to cardiomyocytes.

Do rat cardiac myocytes release ATP on contraction?

ATP is released by numerous cell types in response to mechanical strain. It then acts as a paracrine or autocrine signaling molecule, inducing a variety of biological responses. In this work, we addressed the question whether mechanical force acting on the membranes of contracting cardiomyocytes during periodic longitudinal shortening can stimulate the release of ATP. Electrically stimulated isolated adult rat cardiomyocytes as well as spontaneously contracting mouse cardiomyocytes derived from embryonic stem (ES) cells were assayed for ATP release with the use of luciferase and a sensitive charge-coupled device camera. Sensitivity of soluble luciferase in the supernatant of cardiomyocytes was 100 nM ATP, which is approximately 10-fold below the EC(50) values for most purinergic receptors expressed in the heart (1.5-20 microM). Light intensities were not different between resting or contracting adult rat cardiomyocytes. Similar results were obtained with ES-cell-derived contracting mouse cardiomyocytes. ATP release was measurable only from obviously damaged or permeabilized cells. To increase selectivity and sensitivity of ATP detection we have targeted a recombinant luciferase to the sarcolemmal membrane using a wheat germ agglutinin-IgG linker. Contraction of labeled adult rat cardiomyocytes was not associated with measurable bioluminescence. However, when human umbilical vein endothelial cells were targeted with membrane-bound luciferase, shear stress-induced ATP release could be clearly detected, demonstrating the sensitivity of the detection method. In the present study, we did not detect ATP release from contracting cardiomyocytes on the single cell level, despite adequate sensitivity of the detection system. Thus deformation of the contracting cardiomyocyte is not a key stimulus for the release of cellular ATP.

The Hansenula polymorpha MOX gene presents two alternative transcription start points differentially utilized and sensitive to respiratory activity.

The peroxisomal methanol metabolism of Hansenula polymorpha depends on a group of genes that are coordinately regulated. Methanol oxidase (Mox) plays a key role in this pathway and its synthesis has been shown to be regulated at the transcriptional level. MOX expression is strongly repressed on glucose and activated on glycerol or methanol. In this study we have identified two MOX transcripts that are differentially expressed along MOX derepression. The first one, named l-MOX (for longer MOX), starts at position -425, is only weakly and transiently transcribed and is not translated into the Mox protein. The other is the true MOX mRNA, which initiates around position -25. Using a strain bearing multiple copies of MOX(Q1N) and a reporter gene fused to the MOX promoter, regulation of the two transcripts was investigated. Initiation of the true MOX correlates with repression of l-MOX and conditions that are repressive for MOX transcription, such as the inhibition of mitochondrial activity, lead to higher levels of l-MOX expression. This effect was first observed in a mox mutant (Q1N-M8) unable to grow on nonfermentable carbon sources. No function was detected for l-MOX, but its regulation follows a pattern similar to that of catalase, which is essential for methanol metabolism. This suggests that, l-MOX, although precisely regulated, seems to be a remnant of the evolution of the methanol metabolism network.