Jan Zaujec

Cardiac malformations and myocardial abnormalities in podoplanin knockout mouse embryos: Correlation with abnormal epicardial development.

Epicardium and epicardium‐derived cells have been shown to be necessary for myocardial differentiation. To elucidate the function of podoplanin in epicardial development and myocardial differentiation, we analyzed podoplanin knockout mouse embryos between embryonic day (E) 9.5 and E15.5 using immunohistochemical differentiation markers, morphometry, and three‐dimensional reconstructions. Podoplanin null mice have an increased embryonic lethality, possibly of cardiac origin. Our study reveals impairment in the development of the proepicardial organ, epicardial adhesion, and spreading and migration of the epicardium‐derived cells. Mutant embryos show a hypoplastic and perforated compact and septal myocardium, hypoplastic atrioventricular cushions resulting in atrioventricular valve abnormalities, as well as coronary artery abnormalities. The epicardial pathology is correlated with reduced epithelial–mesenchymal transformation caused by up‐regulation of E‐cadherin, normally down‐regulated by podoplanin. Our results demonstrate a role for podoplanin in normal cardiac development based on epicardial–myocardial interaction. Abnormal epicardial differentiation and reduced epithelial–mesenchymal transformation result in deficient epicardium‐derived cells leading to myocardial pathology and cardiac anomalies. Developmental Dynamics 237:847–857, 2008.

Vascular Endothelial Growth Factor Is Induced by the Inflammatory Cytokines Interleukin-6 and Oncostatin M in Human Adipose Tissue In Vitro and in Murine Adipose Tissue In Vivo

Objectives—It is believed that adipose tissue acts as an endocrine organ by producing inflammatory mediators and thereby contributes to the increased cardiovascular risk seen in obesity. A link between adipose tissue mass and angiogenesis has been suggested. Vascular endothelial growth factor (VEGF) seems to be implicated in this process. Members of the glycoprotein (gp)130 ligand family regulate VEGF expression in other cells. Methods and Results—We used tissue explants as well as primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether the gp130 ligands oncostatin M (OSM), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1) regulate VEGF expression in human adipose tissue. Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in VEGF production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte-differentiation was induced by hormone-supplementation. All cell types responded to IL-6 and OSM with a robust increase in VEGF protein production and a similar increase in VEGF-specific mRNA. Furthermore, IL-1&bgr; synergistically enhanced the effect of OSM on VEGF production. AG-490, a JAK/STAT inhibitor, abolished the OSM-dependent VEGF induction almost completely. In mice, IL-6 and OSM increased serum levels of VEGF and VEGF mRNA and vessel density in adipose tissue. Conclusion—We speculate that the inflammatory cytokines IL-6 and OSM might support angiogenesis during adipose tissue growth by upregulating VEGF.

Indirubin-3′-Monoxime Blocks Vascular Smooth Muscle Cell Proliferation by Inhibition of Signal Transducer and Activator of Transcription 3 Signaling and Reduces Neointima Formation In Vivo

Objective—Our goal was to examine the influence of indirubin-3′-monoxime (I3MO), a natural product–derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. Methods and Results—I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)–BB-induced VSMC proliferation by arresting cells in the G0/G1 phase of the cell cycle as assessed by 5-bromo-2′-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38MAPK was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-&ggr;-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via [14C]l-arginine/[14C]l-citrulline conversion. The specific STAT3 inhibitor Stattic led to decreased VSMC proliferation, and transient expression of a constitutively active form of STAT3 overcame the I3MO-induced cell cycle arrest in mouse embryonic fibroblasts. In a murine femoral artery cuff model, I3MO prevented neointima formation while reducing STAT3 phosphorylation and the amount of proliferating Ki67-positive cells. Conclusion—I3MO represses PDGF- and thrombin-induced VSMC proliferation and, in vivo, neointima formation, likely because it specifically blocks STAT3 signaling. This profile and its positive effect on endothelial NO production turns I3MO into a promising lead compound to prevent restenosis.

Podoplanin deficient mice show a rhoa-related hypoplasia of the sinus venosus myocardium including the sinoatrial node

We investigated the role of podoplanin in development of the sinus venosus myocardium comprising the sinoatrial node, dorsal atrial wall, and primary atrial septum as well as the myocardium of the cardinal and pulmonary veins. We analyzed podoplanin wild‐type and knockout mouse embryos between embryonic day 9.5–15.5 using immunohistochemical marker podoplanin; sinoatrial‐node marker HCN4; myocardial markers MLC‐2a, Nkx2.5, as well as Cx43; coelomic marker WT‐1; and epithelial‐to‐mesenchymal transformation markers E‐cadherin and RhoA. Three‐dimensional reconstructions were made and myocardial morphometry was performed. Podoplanin mutants showed hypoplasia of the sinoatrial node, primary atrial septum, and dorsal atrial wall. Myocardium lining the wall of the cardinal and pulmonary veins was thin and perforated. Impaired myocardial formation is correlated with abnormal epithelial‐to‐mesenchymal transformation of the coelomic epithelium due to up‐regulated E‐cadherin and down‐regulated RhoA, which are controlled by podoplanin. Our results demonstrate an important role for podoplanin in development of sinus venosus myocardium. Developmental Dynamics 238:183–193, 2009.

Pulmonary Vein, Dorsal Atrial Wall and Atrial Septum Abnormalities in Podoplanin Knockout Mice With Disturbed Posterior Heart Field Contribution

The developing sinus venosus myocardium, derived from the posterior heart field, contributes to the atrial septum, the posterior atrial wall, the sino-atrial node, and myocardium lining the pulmonary and cardinal veins, all expressing podoplanin, a coelomic and myocardial marker. We compared development and differentiation of the myocardium and vascular wall of the pulmonary veins (PV), left atrial dorsal wall, and atrial septum in wild type with podoplanin knockout mouse embryos (E10.5–E18.5) by 3D reconstruction and immunohistochemistry. Expression of Nkx2.5 in the pulmonary venous myocardium changes from mosaic to positive during development pointing out a high proliferative rate compared with Nkx2.5 negative myocardium of the sino-atrial node and cardinal veins. In mutants, myocardium of the PVs, dorsal atrial wall and atrial septum was hypoplastic. The atrial septum and right-sided wall of the PV almost lacked interposed mesenchyme. Extension of smooth muscle cells into the left atrial body was diminished. We conclude that myocardium of the PVs, dorsal atrial wall, and atrial septum, as well as the smooth muscle cells, are derived from the posterior heart field regulated by podoplanin.

The inflammatory mediator oncostatin M induces angiopoietin 2 expression in endothelial cells in vitro and in vivo

Summary.  Objectives: Members of the glycoprotein 130 (gp130) receptor–gp130 ligand family play a role in angiogenesis in different tissues. We tested the effect of this cytokine family on the angiopoietin (Ang)–Tie system, which is involved in blood vessel maturation, stabilization, and regression. Results: Oncostatin M (OSM) increased Ang2 expression in human umbilical vein endothelial cells via Janus kinase/signal transducer and activator of transcription (JAK/STAT) and mitogen‐activated protein (MAP) kinase activation. Furthermore, OSM induced Ang2 expression in macrovascular endothelial cells isolated from the human aorta and in microvascular endothelial cells isolated from human heart. Our in vivo experiments revealed that mRNA expression of Ang2 in hearts of mice injected with OSM increased significantly, and levels of OSM mRNA significantly correlated with mRNA levels of Ang2 in human hearts. In addition, OSM increased the expression of its own receptors, gp130 and OSM receptor, in endothelial cells in vitro and in mice in vivo, and levels of OSM mRNA significantly correlated with mRNA levels of gp130 and OSM receptor in human hearts. Conclusion: Our data, showing the effects of OSM on the Ang–Tie system in endothelial cells, in hearts of mice, and in human heart tissue, provide yet another link between inflammation and angiogenesis.

The inflammatory mediator oncostatin M induces stromal derived factor-1 in human adult cardiac cells

Stromal derived factor 1 (SDF‐1) is a CXC chemokine important in the homing process of stem cells to injured tissue. It has been implicated in healing and tissue repair. Growing evidence suggests that the glycoprotein‐130 (gp130) ligand family is involved in repair processes in the heart. The aim of our study was to determine whether gp130 ligands could affect SDF‐1 expression in cardiac cells. Human adult cardiac myocytes (HACMs) and fibroblasts (HACFs) were treated with gp130 ligands. Protein and mRNA levels of SDF‐1 were determined using ELISA and RT‐PCR, respectively. mRNA levels of SDF‐1 were determined in human and mouse heart samples by RT‐PCR. HACMs and HACFs constitutively express SDF‐1, which was significantly up‐regulated by the gp130 ligand oncostatin M (OSM). This effect was counteracted by a p38 inhibitor and to a lesser extent by a PI3K inhibitor. mRNA expression of SDF‐1 in hearts of mice injected with OSM increased significantly. Levels of OSM and SDF‐1 mRNA correlated significantly in human failing hearts. Our data, showing that OSM induces SDF‐1 protein secretion in human cardiac cells in vitro and murine hearts in vivo, suggest that OSM via the induction of SDF‐1 might play a key role in repair and tissue regeneration.— Hohensinner, P. J., Kaun, C., Rychli, K., Niessner, A., Pfaffenberger, S., Rega, G., Furnkranz, A., Uhrin, P., Zaujec, J., Afonyushkin, T., Bochkov, V. N., Maurer, G., Huber, K., Wojta, J. The inflammatory mediator oncostatin M induces stromal derived factor‐1 in human adult cardiac cells. FASEB J. 23, 774–782 (2009)

The interferon stimulated gene 12 inactivates vasculoprotective functions of NR4A nuclear receptors.

Rationale: Innate and adaptive immune responses alter numerous homeostatic processes that are controlled by nuclear hormone receptors. NR4A1 is a nuclear receptor that is induced in vascular pathologies, where it mediates protection. Objective: The underlying mechanisms that regulate the activity of NR4A1 during vascular injury are not clear. We therefore searched for modulators of NR4A1 function that are present during vascular inflammation. Methods and Results: We report that the protein encoded by interferon stimulated gene 12 (ISG12), is a novel interaction partner of NR4A1 that inhibits the transcriptional activities of NR4A1 by mediating its Crm1-dependent nuclear export. Using 2 models of vascular injury, we show that ISG12-deficient mice are protected from neointima formation. This effect is dependent on the presence of NR4A1, as mice deficient for both ISG12 and NR4A1 exhibit neointima formation similar to wild-type mice. Conclusions: These findings identify a previously unrecognized feedback loop activated by interferons that inhibits the vasculoprotective functions of NR4A nuclear receptors, providing a potential new therapeutic target for interferon-driven pathologies.

Thymic medullar conduits-associated podoplanin promotes natural regulatory T cells.

Podoplanin, a mucin-like plasma membrane protein, is expressed by lymphatic endothelial cells and responsible for separation of blood and lymphatic circulation through activation of platelets. Here we show that podoplanin is also expressed by thymic fibroblastic reticular cells (tFRC), a novel thymic medulla stroma cell type associated with thymic conduits, and involved in development of natural regulatory T cells (nTreg). Young mice deficient in podoplanin lack nTreg owing to retardation of CD4(+)CD25(+) thymocytes in the cortex and missing differentiation of Foxp3(+) thymocytes in the medulla. This might be due to CCL21 that delocalizes upon deletion of the CCL21-binding podoplanin from medullar tFRC to cortex areas. The animals do not remain devoid of nTreg but generate them delayed within the first month resulting in Th2-biased hypergammaglobulinemia but not in the death-causing autoimmune phenotype of Foxp3-deficient Scurfy mice.