Martin Müller

Mechanism of the Tumor Necrosis Factor α-mediated Induction of Endothelial Tissue Factor

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo. Transient transfections were performed in bovine aortic endothelial cells to investigate the role of two fundamentally different AP-1 sites and a closely located NF-κB site in the human TF promotor. The NF-κB site is functionally active, since overexpression of NF-κB(p65) resulted in induction of TF mRNA and activity. Promotor analysis showed that NF-κB induction was dependent on the integrity of the region from base pair −188 to −181. Overexpression of Jun/Fos resulted in TF induction of transcription and protein/activity. Functional studies revealed that the proximal AP-1 site, but not the distal, was inducible by Jun/Fos heterodimers. The distal AP-1 site, which has a G → A switch at position 4, was inducible by Jun homodimers. Electrophoretic mobility shift assays, using extracts of tumor necrosis factor α (TNFα)-stimulated bovine aortic endothelial cells, demonstrated TNFα-inducible binding to the proximal AP-1 site, comprising JunD/Fos heterodimers. At the distal AP-1 site, only minor induction of binding activity, characterized as proteins of the Jun and ATF family, was observed. Consistently, this site only marginally participates in TNFα induction. Functional studies with TF promotor plasmids confirmed that deletion of the proximal AP-1 or the NF-κB site decreased TNFα-mediated TF induction to a higher extend than loss of the distal AP-1 site. However, integrity of both AP-1 sites and the NF-κB site was required for optimal TNFα stimulation. The relevance of these in vitro data was confirmed in vivo in a mouse tumor model. Expression plasmids for a dominant negative Jun mutant or I-κB were packaged in liposomes. When either mutated Jun or I-κB were injected intravenously 48 h before TNFα, a reduction in TNFα-mediated TF expression in the tumor endothelial cells was observed. Simultaneously, fibrin/fibrinogen deposition decreased and free blood flow could be restored. Thus, TNFα-induced up-regulation of endothelial cell TF depends on a concerted action of members of the bZIP and NF-κB family.

Insights in Vessel Development and Vascular Disorders Using Targeted Inactivation and Transfer of Vascular Endothelial Growth Factor, the Tissue Factor Receptor, and the Plasminogen System

VEGF has been proposed to participate in normal and pathological vessel formation. Surprisingly, lack of only a single VEGF allele resulted in embryonic lethality due to abnormal formation of intra- and extra-embryonic vessels. Homozygous VEGF-deficient embryos, generated by tetraploid aggregation, revealed an even more severe defect in vessel formation. These results (1) suggest a tight regulation of early vessel development by VEGF and, indirectly, the presence of other VEGF-like molecules; (2) reveal an unprecedented lethal phenotype associated with heterozygous deficiency of an autosomal gene, and (3) demonstrate that tetraploid aggregation was a valid and the only method to study the phenotype of the homozyogous VEGF-deficient embryos. The dominant and strict dose-dependent role of VEGF in vivo renders this molecule a desirable therapeutic target for promoting or preventing angiogenesis. Tissue factor (TF) is the principal cellular initiator of coagulation and its deregulated expression has been related to thrombogenesis in sepsis, cancer, and inflammation. However, TF appears to be also involved in a variety of non-hemostatic functions including inflammation, cancer, brain function, immune response, and tumor-associated angiogenesis. Surprisingly, TF deficiency resulted in embryonic lethality due to abnormal extra-embryonic vessel development and defective vitelloembryonic circulation. The abnormal yolk sac vasculature is reminiscent of that observed in embryos lacking VEGF, possibly suggesting that both gene functions are interconnected. These targeting studies extend the recently documented role of TF in tumor-associated angiogenesis and warrant further study of its role in angiogenesis during other pathological disorders. The plasminogen system, via its triggers, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), has been implicated in thrombosis, arterial neointima formation, and atherosclerosis. Studies in mice with targeted gene inactivation of t-PA, u-PA, PAI-1, the urokinase receptor (u-PAR), and plasminogen (Plg) revealed (1) that deficiency of t-PA or u-PA increase the susceptibility to thrombosis associated with inflammation and that combined deficiency of t-PA:u-PA or deficiency of Plg induces severe spontaneous thrombosis; (2) that vascular injury-induced neointima formation is reduced in mice lacking u-PA-mediated plasmin proteolysis, unaltered in t-PA- or u-PAR-deficient mice and accelerated in PAI-1-deficient mice, but that it can be reverted by adenoviral PAI-1 gene transfer; and (3) that atherosclerosis in mice doubly deficient in apolipoprotein E (apoE) and PAI-1 is reduced after 10 weeks of cholesterol-rich diet. Thus, the plasminogen system significantly affects thrombosis, restenosis, and atherosclerosis.

Loss of caveolin expression in type I pneumocytes as an indicator of subcellular alterations during lung fibrogenesis

Abstract Caveolin is a major structural protein of caveolae, also known as plasmalemmal vesicles, which are particularly abundant in type I pneumocytes and capillary endothelial cells of lung parenchyma. Here we demonstrate that caveolin expression in the alveolar epithelium of rats and mini pigs is strikingly downregulated after irradiation-induced lung injury. Indirect immunoperoxidase staining with polyclonal anti-caveolin antibodies, confirmed by double fluorescence studies with type I cell-specific monoclonal anti-cytokeratin antibodies or lectins, revealed a dramatic loss of caveolin immunoreactivity in type I pneumocytes. In contrast, caveolin expression increased in endothelial cells. Immunoblotting of lung homogenates from normal and irradiated rats using specific anti-caveolin antibodies confirmed the presence of caveolin in normal tissue and its marked decrease of expression in fibrotic tissue. The loss of caveolin as an important structural protein of caveolae in alveolar epithelial cells may be an early indicator of serious type I cell injury during fibrogenesis. The increase of caveolin immunoreactivity in endothelia of blood vessels may indicate that different types of caveolae and/or different regulatory mechanisms of caveolin expression exist.

Differential immunolocalization of VEGF in rat and human adult lung, and in experimental rat lung fibrosis: Light, fluorescence, and electron microscopy

Vascular endothelial growth factor (VEGF) is a cytokine with main angiogenetic functions in embryonic development and tumor‐formation. In the adult lung, reports of the localization of VEGF were controversial. A precise cell typing of VEGF‐positive pulmonary cells is still lacking. Nothing is known about a potential role in pulmonary fibrosis. Immunohistochemistry (IH), double immunofluorescence microscopy (DIF), and immunoelectron microscopy (IEM) were used to study the differential distribution of VEGF in paraffin‐embedded (IH, DIF) and in cryo‐substituted, Lowicryl‐embedded (IEM) specimens of normal rat and human lungs and fibrotic rat lungs. Fibrosis was induced by intratracheal bleomycin treatment. IH and DIF showed that VEGF was present in surfactant protein (SP) D‐positive alveolar type II pneumocytes, bronchiolar Clara cells, smooth muscle (SM) cells, and α‐SM actin‐positive myofibroblasts of normal rat and human lungs. Fibrotic lesions in bleomycin‐treated rat lungs were rich in VEGF‐positive cells presenting with a heterogeneous phenotype (mainly SP‐D‐positive type II pneumocytes, α‐SM actin‐positive myofibroblasts). There were no signs of angiogenesis. Post‐embedding immunogold labeling using protein A‐gold and IgG‐gold technique revealed a specific localization of VEGF to mitochondria, Clara cell secretory granules, and capillary interendothelial cell junctions. The predominant localization of VEGF to bronchiolar and alveolar epithelial and α‐SM actin‐positive cells, and the marked increase of VEGF‐positive type II pneumocytes and myofibroblasts in fibrotic lung lesions, indicate that in adult lungs VEGF is involved in processes other than angiogenesis. Anat Rec 254:61–73, 1999.

Immunohistochemical detection of tissue factor (TF) on paraffin sections of routinely fixed human tissue

Tissue factor (TF), a 47 kDa transmembrane glycoprotein, is the essential receptor and cofactor for factor VII/VIIa. Its distribution in normal tissues and in tumours has been recently investigated immunohistochemically with monoclonal and polyclonal anti-TF antibodies in frozen sections. The cardinal problem of this technique is the difficulty of determining exactly the localization of the reaction product at least in certain tissues. Here, we demonstrate a method using monoclonal anti-TF antibodies to detect TF in routinely fixed, microwaved, paraffin-embedded tissues. Generally, there were no fundamental differences in TF distribution in frozen and paraffin-embedded material. However, in most cases, the paraffin sections allow a better cellular localization of TF. For example, the staining pattern for TF in both kinds of sections is identical in kidney, brain and skin. The paraffin-embedded material, however, clearly shows that TF expression is restricted to the parietal and the visceral epithelia of Bowmans capsule of glomeruli in the kidney, and to astrocytes and their processes in the brain. TF reactivity in the skin is revealed to be cell membrane-bound; in cardiomyocytes TF shows an exclusively sarcolemmal localization. The immunohistological detection of TF in paraffin sections is a powerful tool for systematic studies on the possible role of TF in the context of physiological and pathological studies.

NFATc1 affects mouse splenic B cell function by controlling the calcineurin--NFAT signaling network.

Mouse B cells lacking NFATc1 exhibit defective proliferation, survival, isotype class switching, cytokine production, and T cell help.

Distribution of E-cadherin and Ep-CAM in the human lung during development and after injury

Paraffin sections were obtained of human fetal, adult, and pathological lung (pulmonary fibrosis after radiotherapy or chemotherapy). The localization of epithelial adhesion molecules E-cadherin and Ep-CAM (former epithelial surface 40 kDa glycoprotein) was investigated by immunoperoxidase and/or immunofluorescence techniques with monoclonal antibodies. During development, the epithelia of the primary pulmonary primordium, the secondary bronchi and the adult bronchial epithelium retained immunoreactivity for E-cadherin and Ep-CAM with lateral immunostaining of cell membranes. In normal adult lungs, Ep-CAM was detected in type I and II alveolar epithelial cells, whereas E-cadherin was confined to the basolateral domain of type II cells. In pulmonary fibrosis, Ep-CAM could be further detected on the cell surface of epithelial remnants. In contrast, E-cadherin expression was characterized by a change of the membrane localization to a spotty, cytoplasmic pattern in the alveolar epithelium, possibly indicating functional inactivation of the protein during fibrogenesis.

Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development

NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least 3 different kinases: CK1, GSK-3, and DYRK. The major docking sites for calcineurin and CK1 are strongly conserved throughout vertebrate evolution, and conversion of either the calcineurin docking site to a high-affinity version or the CK1 docking site to a low-affinity version results in generation of hyperactivable NFAT proteins that are still fully responsive to stimulation. In this study, we generated transgenic mice expressing hyperactivable versions of NFAT1 from the ROSA26 locus. We show that hyperactivable NFAT increases the expression of NFAT-dependent cytokines by differentiated T cells as expected, but exerts unexpected signal-dependent effects during T cell differentiation in the thymus, and is progressively deleterious for the development of B cells from hematopoietic stem cells. Moreover, progressively hyperactivable versions of NFAT1 are increasingly deleterious for embryonic development, particularly when normal embryos are also present in utero. Forced expression of hyperactivable NFAT1 in the developing embryo leads to mosaic expression in many tissues, and the hyperactivable proteins are barely tolerated in organs such as brain, and cardiac and skeletal muscle. Our results highlight the need for balanced Ca/NFAT signaling in hematopoietic stem cells and progenitor cells of the developing embryo, and emphasize the evolutionary importance of kinase and phosphatase docking sites in preventing inappropriate activation of NFAT.

Upregulation of gap junction protein connexin43 in alveolar epithelial cells of rats with radiation-induced pulmonary fibrosis

The degree of immunoreactive connexin43 (Cx43) in rat lung was evaluated during the development of radiation-induced pulmonary fibrosis in rat by a double immunofluorescence technique using polyclonal antisera of Cx43 and monoclonal antibodies to cytokeratins on cryostat sections. In normal rat lungs, Cx43 was detected in pneumocytes type II and I, in large blood vessel endothelia, in peribronchial smooth muscle cells, and in some peribronchial and perivascular interstitial cells. As early as 1 week after irradiation, enhanced immunoreactivity for Cx43 in the epithelial cells was detected. In severely injured lungs (about 3 months after irradiation), Cx43 was found also in the cytoplasm of type II pneumocytes. These findings were confirmed by western blot data. Western blot analysis also revealed increased phosphorylation of Cx43. It remains to be investigated whether the increased content of Cx43 in irradiated rat lung may be due to an enhanced number of gap junction between type I and II alveolar epithelial cells.

Functional implications of tissue factor localization to cell-cell contacts in myocardium.

Recently published studies suggest that the procoagulant receptor protein tissue factor (TF) is involved in vitro in cell adhesion and migration, via an interaction of its cytoplasmic domain with cytoskeletal proteins. Interestingly, TF is abundantly expressed in myocardium, but not in skeletal muscle. To elucidate the possible roles of TF in the myocardium, this study examined the cellular distribution of TF in relation to cytoskeletal proteins, as well as its relative amounts in different segments of premature, mature, and pathologically altered cardiac muscle. In juvenile and adult hearts, TF was predominantly detectable in the transverse part of the intercalated discs, where it co‐localized with cytoskeletal proteins such as desmin and vinculin. The lowest amount of TF was observed in right atrial and the highest in left ventricular myocardium, which correlated with the number of contact sites of cardiomyocytes in these segments of the cardiac muscle. Lower levels of TF were present in structurally altered myocardium from patients with hypertension or ventricular hypertrophy. In addition, TF expression was decreased in human heart during sepsis and transiently decreased in rabbit heart in an endotoxaemia model, which indicates that a reduction in TF may contribute to cardiac failure in sepsis. The microtopography of TF at cardiomyocyte contact sites indicates that TF may play a structural role in the maintenance of cardiac muscle. Copyright