Albrecht Lepple-Wienhues

A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF‐E. VEGF‐E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF‐E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat‐stable, secreted dimer. VEGF‐E and VEGF‐A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF‐A, VEGF‐E was found to bind with high affinity to VEGF receptor‐2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF‐A, VEGF‐E did not bind to VEGF receptor‐1 (Flt‐1). VEGF‐E is thus a potent angiogenic factor selectively binding to VEGF receptor‐2. These data strongly indicate that activation of VEGF receptor‐2 alone can efficiently stimulate angiogenesis.

Cell volume in the regulation of cell proliferation and apoptotic cell death.

Cell proliferation must – at some time point – lead to increase of cell volume and one of the hallmarks of apoptosis is cell shrinkage. At constant extracellular osmolarity those alterations of cell volume must reflect respective changes of cellular osmolarity which are hardly possible without the participation of cell volume regulatory mechanisms. Indeed, as shown for ras oncogene expressing 3T3 fibroblasts, cell proliferation is paralleled by activation of Na+/H+ exchange and Na+,K+,2Cl- cotransport, the major transport systems accomplishing regulatory cell volume increase. Conversely, as evident from CD95-induced apoptotic cell death, apoptosis is paralleled by inhibition of Na+/H+ exchanger and by activation of Cl- channels and release of the organic osmolyte taurine, major components of regulatory cell volume decrease. However, ras oncogene activation leads to activation and CD95 receptor triggering to inhibition of K+ channels. The effects counteract the respective cell volume changes. Presumably, they serve to regulate cell membrane potential, which is decisive for Ca++ entry through ICRAC and the generation of cytosolic Ca++ oscillations in proliferating cells. As a matter of fact ICRAC is activated in ras oncogene expressing cells and inhibited in CD95-triggered cells. Activation of K+ channels and Na+/H+ exchanger as well as Ca++ oscillations have been observed in a wide variety of cells upon exposure to diverse mitogenic factors. Conversely, diverse apoptotic factors have been shown to activate Cl- channels and organic osmolyte release. Inhibition of K+ channels is apparently, however, not a constant phenomenon paralleling apoptosis which in some cells may even require the operation of K+ channels. Moreover, cell proliferation may at some point require activation of Cl- channels. In any case, the alterations of cell volume are obviously important for the outcome, as cell shrinkage impedes cell proliferation and apoptosis can be elicited by increase of extracellular osmolarity. At this stage little is known about the interplay of cell volume regulatory mechanisms and the cellular machinery leading to mitosis or death of the cell. Thus, considerable further experimental effort is required in this exciting area of cell physiology.

Impact of localized radiotherapy on blood immune cells counts and function in humans.

Immune cells subsets were prospectively analyzed after localized radiotherapy (LRT). LRT reduced the levels of all lymphocyte subsets, with B-cells and naive T-cells being most sensitive. Lymphocyte function was suppressed, but still within the normal range. Rapid recovery of cytotoxic T-cells/natural killer cells after LRT and the functional suppression within normal levels explains the low incidence of infections after LRT.

Tyrosine Kinases Open Lymphocyte Chloride Channels

Osmotic swelling of lymphocytes opens outwardly rectifying Cl- channels (ORCC) through the src-like kinase p56lck. The central role of this tyrosine protein kinase has been shown by genetic and pharmacologic manipulation of the enzyme. Furthermore, p56lck activates ORCC independently of cell volume increase. ORCC in lymphocytes and epithelial cells from cystic fibrosis (CF) patients are resistant to activation by cAMP. However, osmotic swelling as well as intracellular purified p56lck can activate ORCC in CF lymphocytes. In non-CF lymphocytes ORCC is opened by either, intracellular cAMP, p56lck or by osmotic swelling. Osmotic activation of ORCC can be blocked by the tyrosine kinase inhibitor lavendustin in both cell types. Regulation of ORCC by p56lck thus represents an alternative pathway of stimulating membrane chloride conductance that is left functional in cystic fibrosis. In addition to osmoregulation these mechanisms could play a major role when cells actively change their volume, i.e. during proliferation and apoptosis. Activation of the tyrosine kinase p56lck is an important regulatory step for opening of chloride channels in lymphocytes.

Intracellular mechanisms of l-selectin induced capping

Leucocyte adhesion to endothelial cells is a tightly regulated process involving selectins, integrins and immunoglobulin-like proteins. Cell adhesion and communication are controlled by membrane dynamics like receptor capping. Capping of surface receptors is an ubiquitous mechanism but still not well understood. Employing immunofluorescence techniques, we demonstrate that L-selectin triggering results in receptor capping of the L-selectin molecules in lymphocytes. Using pharmacological inhibitors and genetic deficient cell lines we show that this process involves intracellular signalling molecules. L-Selectin capping seems to be independent on activation of p56lck-kinase, but requires the neutral sphingomyelinase, small G proteins and the cytoskeleton. Therefore, capping of L-selectin upon stimulation might play an important role in the very early phase of lymphocyte trafficking.

Cell volume regulatory mechanisms in apoptotic cell death.

One of the hallmarks of apoptosis is cell shrinkage, which — at constant extracellular osmolarity — requires a decrease of cellular osmolarity. Moreover, apoptosis can be elicited by increase of extracellular osmolarity and the resistance of cells towards apoptosis correlates with their ability to regulate their volume in hypertonic environment. On the other hand, CD95-receptor-mediated apoptosis is blunted at moderate increases of extracellular osmolarity. Given the role of cell volume alterations it is not surprising that apoptosis is paralleled by marked alterations of cell volume regulatory mechanisms. Stimulation of the CD95-receptor, which confers apoptosis to a variety of cells, leads to activation of cell volume regulatory anion channel ORCC. However, activation of ORCC is paralleled by inhibition of cell volume regulatory K+ channel Kv1.3. It is only 40 to 60 minutes after triggering of the CD95-receptor when the cells release the organic osmolyte taurine and shrink.ZusammenfassungEines der charakteristischen Kennzeichen von Apoptose ist Zellschrumpfung, die eine Abnahme der zellulären Osmolarität erfordert. Dies ist ohne Beteiligung zellvolumenregulierender Mechanismen nicht möglich. Bei der Zellvolumenregulation können die Zellen durch Elektrolytaufnahme über Na+/H+-Austauscher, Na+, K+, 2Cl−-Kotransport und Na+-Kanäle schwellen sowie durch Elektrolytverlust über Ionenkanäle (K+-Kanäle und Cl−-Kanäle) und KCl-Symport schrumpfen. Darüber hinaus können Zellen durch Aufnahme oder Bildung von organischen Osmolyten (unter anderem Sorbitol, Inositol, Betain, Glycerophosphorylcholin und Taurin) ihre Osmolarität und damit ihr Volumen steigern und durch Osmolytabgabe ihre Osmolarität senken. Apoptose kann durch massive osmotische Zellschrumpfung ausgelöst werden, und die Fähigkeit von Zellen, einen hyperosmolaren Schock ohne Apoptose zu überstehen, korreliert mit ihrer Fähigkeit zur Zellvolumenregulation. Die CD95-Rezeptorvermittelte Apoptose von Jurkat-T-Lymphozyten wird andererseits durch moderate osmotische Zellschrumpfung gehemmt. Angesichts der Bedeutung des Zellvolumens für die Apoptose überrascht es nicht, daß die rezeptorvermittelte Apoptose von veränderter Aktivität zellvolumenregulierter Transportprozesse an der Zellmembran begleitet ist. Stimulation des CD95-Rezeptors führt zur Aktivierung der zellvolumenregulierten Anionenkanäle OrCC (outwardly rectifying chloride channels), jedoch gleichzeitig zur Hemmung der zellvolumenregulierten K+-Kanäle Kv1.3. Erst etwa 60 Minuten nach Aktivierung des CD95-Rezeptors kommt es zur Zellschrumpfung. Dabei eliminieren die Zellen den organischen Osmolyten Taurin.

Effect of Sphingosine on Ca2+ Entry and Mitochondrial Potential of Jurkat T Cells - Interaction with Bcl2

Triggers of Jurkat T cell apoptosis include sphingosine and ceramide. Sphingosine and ceramide further inhibit capacitative Ca2+ entry (ICRAC), an effect leading to inactivation but not death of Jurkat T cells. Mitochondria are key organelles in the machinery leading to apoptosis and on the other hand have been shown to participate in the regulation of Ca2+ entry. The present experiments were performed to explore whether treatment of Jurkat T cells with sphingosine leads to apoptosis and reduced Ca2+ entry and whether those effects are sensitive to expression of the antiapoptotic protein Bcl2, localized in the outer mitochondrial membrane. Exposure of Jurkat T cells to 10 µM spingosine was according to DiOC6 fluorescence followed by mitochondrial depolarization and according to Fura-red/Fluo-3 fluorescence followed by decreased capacitative Ca2+ entry. Mitochondrial depolarization was significantly delayed in cells overexpressing wild type Bcl2 or Bcl2 targeted to the mitochondrial membrane, whereas no significant influence on mitochondrial depolarization was observed in cells expressing Bcl2 lacking the membrane targeting motif or Bcl2 targeted to the endoplasmatic reticulum. In contrast to mitochondrial potential, the blunting of capacitative Ca2+ entry following sphingosine treatment was not sensitive to mitochondrial Bcl2 expression. In conclusion sphingosine exposure leads to both, mitochondrial depolarization and inhibition of capacitative Ca2+ entry. Mitochondrial Bcl2 reverses the effect on mitochondria but not on Ca2+ entry and thus leads to dissociation of those two sequelae of sphingosine treatment.

A Mysterious Channel: Properties of the Capacitive Ca2+ Channel in Lymphocytes

A Ca2+ current operated by the filling state of intracellular Ca2+ stores was first described in T cells. This store-operated current (SOC) was later named CRAC (Ca2+