Antje Augstein

Cell-specific and hypoxia-dependent regulation of human HIF-3α: inhibition of the expression of HIF target genes in vascular cells

Hypoxia-inducible factors (HIF) are transcription factors responding to reduced oxygen levels and are of utmost importance for regulation of a widespread of cellular processes, e.g., angiogenesis. In contrast to HIF-1α/HIF-2α, the relevance of HIF-3α for the regulation of the HIF pathway in human vascular cells is largely unknown. HIF-3α mRNA increases under hypoxia in endothelial and vascular smooth muscle cells. Analysis of HIF-3α isoforms revealed a cell type-specific pattern, but only one isoform, HIF-3α2, is hypoxia-inducible. Reporter gene assays of the appropriate promoter localized a 31-bp fragment, mediating this hypoxic regulation. The contribution of HIF-1/2 and NFκB to the HIF-3α induction was verified. Functional studies focused on overexpression of HIF-3α isoforms, which decrease the hypoxia-mediated expression of VEGFA and Enolase2. These data support the notion of a hypoxia-induced inhibitory function of HIF-3α and demonstrate for the first time the existence of this negative regulation of HIF-signaling in vascular cells.

Ephrin-A1/EphA4-mediated adhesion of monocytes to endothelial cells

The Eph receptors represent the largest family of receptor tyrosine kinases. Both Eph receptors and their ephrin ligands are cell-surface proteins, and they typically mediate cell-to-cell communication by interacting at sites of intercellular contact. The major aim of the present study was to investigate the involvement of EphA4-ephrin-A1 interaction in monocyte adhesion to endothelial cells, as this process is a crucial step during the initiation and progression of the atherosclerotic plaque. Immunohistochemical analysis of human atherosclerotic plaques revealed expression of EphA4 receptor and ephrin-A1 ligand in major cell types within the plaque. Short-time stimulation of endothelial cells with the soluble ligand ephrin-A1 leads to a fourfold increase in adhesion of human monocytes to endothelial cells. In addition, ephrin-A1 further increases monocyte adhesion to already inflamed endothelial cells. EphrinA1 mediates its effect on monocyte adhesion via the activated receptor EphA4. This ephrinA1/EphA4 induced process involves the activation of the Rho signaling pathway and does not require active transcription. Rho activation downstream of EphA4 leads to increased polymerization of actin filaments in endothelial cells. This process was shown to be crucial for the proadhesive effect of ephrin-A1. The results of the present study show that ephrin-A1-induced EphA4 forward signaling promotes monocyte adhesion to endothelial cells via activation of RhoA and subsequent stress-fiber formation by a non-transcriptional mechanism.

Bleomycin induces IL-8 and ICAM-1 expression in microvascular pulmonary endothelial cells.

To investigate the pathomechanisms of bleomycin-induced early inflammation of lung parenchyma which is known to result in pulmonary fibrosis, we examined the in vitro effect of bleomycin (BLM) on primary human pulmonary microvascular endothelial cells (HMVEC-L). After incubation of microvascular endothelial cells with BLM we detected an induced phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) by immunoblotting. Further, after BLM-exposure an increased concentration of interleukin-8 (IL-8) in culture supernatant and an increased expression of intercellular adhesion molecule-1 (ICAM-1, CD54) on the cell surface have been observed. Real-time PCR revealed up-regulated mRNA expression levels of both, IL-8 and ICAM-1 after treatment with BLM. Finally, pre-treatment with a selective p38 MAPK-inhibitor, SB 203580, potently reduced the BLM-induced up-regulation of IL-8 expression but did not show any effect on expression of ICAM-1. These results demonstrate that BLM induces the expression of pro-inflammatory molecules in the pulmonary microvascular endothelium, which thereby may actively contribute to the development of early inflammation and later fibrosis of the lung. Furthermore, investigating the effect of an inhibitor of p38 MAPK the data indicate the involvement of p38 MAPK-dependent as well as p38 MAPK-independent mechanisms in the effects of BLM on the pulmonary microvasculature.

OxLDL and macrophage survival: essential and oxygen-independent involvement of the Hif-pathway

Atherosclerotic plaques are characterized by hypoxic even anoxic areas and by high concentrations of oxidized lipoproteins. Moreover, unstable plaques attract a high number of macrophages despite the proapoptotic background within these plaques. Recently, it was shown that these macrophages are positive for Hif-1α. This subunit is a part of hypoxia-inducible factor 1 (Hif-1), a key transcriptional factor under hypoxia. Till date, it is not understood whether the Hif-system (consisting of Hif-1, Hif-2 and Hif-3) is involved in protection of macrophages under these proatherogenic conditions. The present study delineates that oxLDL causes fundamental changes in the regulation of the Hif-system in primary human macrophages. First, both oxLDL and hypoxia mediate accumulation of Hif-1α protein. Second, treatment with a combination of oxLDL and hypoxia is acting in an additive manner on Hif-1α protein content. Third, oxLDL alone does not increase Hif-2α protein, but abolishes the hypoxic induction of Hif-2α completely. OxLDL treatment alone was not toxic for macrophages under neither normoxia nor hypoxia. But, inhibition of Hif-pathway by adenoviral expression of a dominant-negative mutant combined with oxLDL treatment independently of the oxygen tension leads to apoptosis, as determined by caspase-3 activation and induction of DNA fragmentation. Furthermore, this inhibition also mediates the opening of the mitochondrial permeability transition pore. In conclusion, the present data show that Hif-1α regulation is essential for survival of oxLDL-treated macrophages independent of the oxygen tension. Therefore, this newly characterized mechanism might also have an important influence for the vulnerability of atherosclerotic plaques.

trans-dominant mutations in the GPR1 gene cause high sensitivity to acetic acid and ethanol in the yeast Yarrowia lipolytica.

Acetate non‐utilizing strains harbouring trans‐dominant mutations in the GPR1 gene (GPR1d) of the dimorphic yeast Yarrowia lipolytica have been selected and characterized. These mutants are highly sensitive to low concentrations of acetic acid and ethanol, even in presence of glucose. The toxic effect of acetic acid is pH‐dependent and has the strongest effect at low pH. In contrast, the action of ethanol is pH‐independent. One GPR1d mutant has been detected that was highly sensitive to acetic acid but could still grow on ethanol, which indicates putative differences in the function of the GPR1 gene product in the sensitivity to acetic acid and ethanol. The GPR1d mutants exhibit a complex pleiotropic phenotype. The mutations cause changed colony morphology as well as dimorphism of cells, and induce early cell death during growth on glucose, even without the presence of dicarbon compounds. Composition of intracellular membranes, as well as morphology of vacuole and mitochondria, were strongly changed. Back‐crosses with wild‐type strains and analysis of recombinant strains have shown that the expression of the pleiotropic phenotype depends on the site of mutation in the GPR1 gene, as well as on the genetic background of the strain harbouring the responsive mutation. Our data suggest that Gpr1p is involved in a general response of cells to the toxic action of dicarbon compounds like acetic acid and ethanol. Copyright

Regulation of the HIF-system in human macrophages--differential regulation of HIF-α subunits under sustained hypoxia.

Macrophages are often associated to pathophysiological processes and were found at hypoxic areas. However, cell adaption greatly depends on hypoxia-inducible factors (HIF). Activation of these transcription factors is induced by heterodimerization of an α-(HIF-1α, -2α, -3α) and HIF-1β subunit. The main regulatory pathway is represented by α-subunit stability. Beside, little is known about the exact mechanisms of fine-tuning in Hif-regulation. The present study characterizes the hypoxia-induced regulation of HIF-1α and -2α in human macrophages. The hypoxic increase of both subunits is initially mediated by protein stabilization. Sustained hypoxia caused a distinct regulation of HIF-1α and -2α. The striking increase of HIF-2α protein expression was contrasted by a dramatic decrease of HIF-1α. The long-term downregulation of HIF-1α is due to downregulation of its mRNA. This decrease was accompanied by increased expression of ahif, a natural cis-antisense transcript of HIF-1α. The ahif-transcript was strongly inducible by hypoxia and rapidly degraded under reoxygenation. Using an adenoviral overexpression and siRNA silencing approach revealed that the targeted regulation of ahif is mediated by the HIF-system itself. Furthermore it could be shown that ahif indeed is able to modulate the hypoxic expression of HIF-1α and influences the expression of the HIF-target gene Enolase-2. Taken together, this study characterizes a new regulation process of the HIF-transcription factor-system in human macrophages under hypoxia. For the first time evidence is provided that ahif is regulated by the HIF-system and influences HIF-1α expression in primary human macrophages.

TNF-α-mediated adhesion of monocytes to endothelial cells—The role of ephrinA1

The ligand ephrin A1 is more often discussed to play a role in the development of the atherosclerotic plaque and in this context especially in the monocyte adhesion to endothelial cells. As tumor necrosis factor-α (TNF-α) is known to induce monocyte adhesion to endothelium and ephrin A1 expression, the present study focuses on the involvement of ephrin A1 in TNF-α-mediated monocyte adhesion. The analysis of different members of the Eph/ephrin system in TNF-α-treated human umbilical vein endothelial cells (HUVEC) revealed that especially ephrinA1 was found to be highly regulated by TNF-α compared to other members of the Eph family. This effect is also present in arterial endothelial cells from the umbilical artery and from the coronary artery. This regulation is dependent on NFκB-activation as shown by the expression of a constitutive-active IκB-mutant. By using siRNA-mediated silencing and adenoviral overexpression of ephrinA1 in HUVEC, the involvement of ephrinA1 in the TNF-α triggered monocyte adhesion to endothelial cells could be demonstrated. In addition, these results could be verified by quantitative adhesion measurement using atomic force microscopy-based single-cell force spectroscopy and under flow conditions. Furthermore, this effect is mediated via the EphA4 receptor. EphrinA1 does not influence the mRNA or protein expression of the adhesion receptors VCAM-1 and ICAM-1 in endothelial cells. However, the surface presentation of these adhesion receptors is modulated in an ephrinA1-dependent manner. In conclusion, these data demonstrate that ephrinA1 plays an important role in the TNF-α-mediated adhesion of monocytes to endothelial cells, which might be of great importance in the context of atherosclerosis.

Local inhibition of hypoxia-inducible factor reduces neointima formation after arterial injury in ApoE-/- mice.

OBJECTIVE Hypoxia plays a pivotal role in development and progression of restenosis after vascular injury. Under hypoxic conditions the hypoxia-inducible factors (HIFs) are the most important transcription factors for the adaption to reduced oxygen supply. Therefore the aim of the study was to investigate the effect of a local HIF-inhibition and overexpression on atherosclerotic plaque development in a murine vascular injury model. METHODS AND RESULTS After wire-induced vascular injury in ApoE-/- mice a transient, local inhibition of HIF as well as an overexpression approach of the different HIF-subunits (HIF-1α, HIF-2α) by adenoviral infection was performed. The local inhibition of the HIF-pathway using a dominant-negative mutant dramatically reduced the extent of neointima formation. The diminished plaque size was associated with decreased expression of the well-known HIF-target genes vascular endothelial growth factor-A (VEGF-A) and its receptors Flt-1 and Flk-1. In contrast, the local overexpression of HIF-1α and HIF-2α further increased the plaque size after wire-induced vascular injury. CONCLUSIONS Local HIF-inhibition decreases and HIF-α overexpression increases the injury induced neointima formation. These findings provide new insight into the pathogenesis of atherosclerosis and may lead to new therapeutic options for the treatment of in stent restenosis.

Effect of P2X7 receptor knockout on AQP-5 expression of type I alveolar epithelial cells.

P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis.

EphrinB2/EphA4-mediated activation of endothelial cells increases monocyte adhesion

The membrane anchored ligand ephrinB2 belongs to the broad Eph/ephrin system and is able to activate different Eph receptors. The Eph receptors belong to the huge group of receptor-tyrosine kinases. Eph receptors as well as their corresponding ephrin ligands are cell-membrane attached proteins. Therefore, direct cell-cell contact is essentially for interaction. It is known that ephrinB2 plays a pivotal role in developmental and in tumour angiogenesis. Previous studies point to a crucial role of the EphA4-receptor in the process of monocyte adhesion. Since ephrinB2 is known as an interaction partner of EphA4, the aim of the present study was to investigate a possible interplay of EphA4-receptor with ephrinB2 during monocyte adhesion to the endothelium. As verified by bulk adhesion assays and atomic-force microscopy based single-cell force spectroscopy, temporary stimulation of endothelial cells from different sources with the soluble ligand ephrinB2 increased monocyte adhesion to endothelial cells. The proadhesive effect of ephrinB2 was independent of an active transcription, but is mediated via the Rho signaling pathway with subsequent modulation of the actin cytoskeleton. Furthermore, ephrinB2 mediated its impact on monocyte adhesion via the receptor EphA4 as shown by siRNA-mediated silencing. Interestingly, ephrinB2 was induced by TNF-α treatment. Silencing of ephrinB2 led to a lowering of the TNF-α mediated monocyte adhesion to endothelial cells. Furthermore, immunohistochemical staining of human atherosclerotic plaque revealed expression of ephrinB2 in macrophages. The results of the present study point to a crucial role of ephrinB2 induced EphA4 forward signaling in the context of monocyte adhesion to endothelial cells. This transcription-independent effect is mediated by Rho signaling induced actin-filament polymerization.