Gottfried Dohr

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

Fas and Fas-Ligand Are Expressed in the Uteroplacental Unit of First-Trimester Pregnancy

PROBLEM: Fas and Fas‐ligand (FasL) are thought to provide a strategy for reducing graft rejection in immunologically ‘privileged’ tissues by controlling injurious lymphocyte reactions. As the uteroplacental unit is often defined as an immune‐privileged site, we investigated the expression of Fas and FasL in this tissue in the first trimester of pregnancy.

Amnion Epithelial Cells, in Contrast to Trophoblast Cells, Express All Classical HLA Class I Molecules Together With HLA-G

PROBLEM: The expression of the non‐classical HLA‐G gene has been shown at the protein level on trophoblast‐derived embryonic tissue, like the extravillous cytotrophoblast. However, the presence of HLA‐G on embryoblast‐derived cells is currently controversial. The amnion epithelium is an embryoblast‐derived cell layer covering the amnion cavity and is the main source for the amnion fluid.

Expression of HLA class I molecules in human first trimester and term placenta trophoblast

Abstract.Expression of HLA class I molecules in trophoblast cells from various locations in normal human first trimester and term placenta was investigated by immunohistochemistry with a panel of monoclonal antibodies against the heavy chains or complete HLA class I molecules complexed with β2-microglobulin. These reagents were also employed to distinguish between the products of different HLA class I loci. In addition to previously characterized reagents, a novel monoclonal antibody against HLA-A molecules (TÜ155) was used. Various choriocarcinoma and transfected cell lines served as controls for the specificities of the monoclonal antibodies. Cells in close contact with maternal cells, such as invading trophoblast cells and cells of the basal plate, expressed β2-m micro globulin in association with HLA-G and HLA-C heavy chains. These class I heavy chains may also have been present as isolated molecules, although not in each of the cells. In contrast, cells of the chorion laeve exclusively expressed HLA-G, and not HLA-A, -B, or -C antigens. Our data support the often discussed immune protective function and the regulatory function of the HLA-G molecule, during invasion. In addition, by using monoclonal antibodies HCA2 (anti-HLA-A and -G), HC10 (anti-HLA-B and -C), TÜ149 (anti-HLA-B, -C, and some -A alleles), SFR8-B6 (anti-HLA-Bw6 and some -C), LA45 (some HLA-A and -B), TÜ48 (anti-HLA-Bw4 and some -A), and TÜ155 (anti-HLA-A), we show the presence of HLA-C molecules in all extravillous trophoblast cells of the cell columns and in the basal plate; the trophoblast cells of the chorion laeve lack this antigen. The function of this molecule is not clear, although a protective function against natural killer cell activity in the endometrium is postulated.

Prooxidant activity of melatonin promotes fas-induced cell death in human leukemic Jurkat cells.

The antioxidant activity of melatonin (MEL) has been considered to constitute part of its physiological as well as pharmacological effects. However, as described herein we found a profound prooxidant activity of micro‐ to millimolar concentrations of MEL in the human leukemic Jurkat cell line. This prooxidant effect was increased in glutathione‐depleted cells and counteracted by antioxidants. As a consequence MEL promoted fas‐induced cell death. These data therefore indicate that MEL may be a modulator of the cellular redox status, but does not necessarily act as an intracellular antioxidant.

Generation, Characterization, and Histochemical Application of Monoclonal Antibodies Selectively Recognizing Oxidatively Modified ApoB-Containing Serum Lipoproteins

To investigate either the role oxidized LDL plays in atherosclerosis or structural changes on the surface of oxidized LDL, monoclonal antibodies (mAbs) are an important tool. After immunizing mice with Cu(2+)-oxidized LDL (oxLDL) and fusion of splenocytes, hybridoma supernatants were screened and cloned. Two mAbs, OB/04 and OB/09 (IgG and IgM), were further characterized. In solid-phase fluorescence immunoassays and Western blot analysis both mAbs reacted with oxLDL, LDL oxidized by a free radical-generating azo compound, or oxVLDL but not with native LDL, acetylated LDL, oxHDL3, azo-oxidized HDL3, or HDL3 modified with malondialdehyde (MDA). In competitive immunoassays with LDL modified by oxidized fatty acid-derived aldehydes, mAb OB/09 strongly reacted with MDA-LDL or MDA-VLDL and LDL modified with 4-hydroxyhexenal followed by 4-hydroxynonenal but not with 4-hydroxyoctenal or hepta-2,4-dienal. mAb OB/04 had a weak affinity for LDL after modification with these aldehydes except for MDA-LDL. LDL modified with arachidonic acid oxidation products (AAOPs) was also recognized by this mAb. However, albumin modified either by the aldehydes applied or by AAOPs did not react with either mAb. Thus, the data indicate that each of the mAbs recognizes a different epitope that is expressed only on apoB-containing lipoproteins upon oxidative modification. An immunostaining with mAb OB/04 was obtained in areas rich in macrophages and in connective tissue of a human atherosclerotic lesion.

Embryonic Stem Cells: Similarities and Differences Between Human and Murine Embryonic Stem Cells

The derivation of murine embryonic stem (mES) cell lines was reported for the first time in 1981 (Nature, 1981; 292:154–156; Proc Natl Acad Sci U S A, 1981; 78:7634–7638), and they have since proved to be a very useful tool with which to study mammalian development, which is characterized by pluripotency and differentiation. About 20 years later, the successful generation of human embryonic stem (hES) cell lines was described (Science, 1998; 282:1145–1147). Although mES and hES are derived from mammals, they cannot be looked at as being one and the same. While basic information for hES can be derived from mES, such information does not correspond on a one‐to‐one basis. This review gives an overview of the characteristics of embryonic stem cells with the main focus on the similarities and differences between human and mES cells.

Macrophages of Human First Trimester Decidua Express Markers Associated to Alternative Activation

Problem:  Depending on the type of their activation, macrophages may promote TH1‐ or TH2‐type of immune responses. To date, not much is known about the activation phenotype of decidua macrophages, which – together with NK cells – constitute the majority of bone marrow derived cells at this location.

Human fetal placental endothelial cells have a mature arterial and a juvenile venous phenotype with adipogenic and osteogenic differentiation potential

Growing interest in the sources of origin of blood vessel related diseases has led to an increasing knowledge about the heterogeneity and plasticity of endothelial cells lining arteries and veins. So far, most of these studies were performed on animal models. Here, we hypothesized that the plasticity of human fetal endothelial cells depends on their vascular bed of origin i.e. vein or artery and further that the differences between arterial and venous endothelial cells would extend to phenotype and genotype. We established a method for the isolation of fetal arterial and venous endothelial cells from the human placenta and studied the characteristics of both cell types. Human placental arterial endothelial cells (HPAEC) and human placental venous endothelial cells (HPVEC) express classical endothelial markers and differ in their phenotypic, genotypic, and functional characteristics: HPAEC are polygonal cells with a smooth surface growing in loose arrangements and forming monolayers with classical endothelial cobblestone morphology. They express artery-related genes (hey-2, connexin 40, depp) and more endothelial-associated genes than HPVEC. Functional testing demonstrated that vascular endothelial growth factors (VEGFs) induce a higher proliferative response on HPAEC, whereas placental growth factors (PlGFs) are only effective on HPVEC. HPVEC are spindle-shaped cells with numerous microvilli at their surface. They grow closely apposed to each other, form fibroblastoid swirling patterns at confluence and have shorter generation and population doubling times than HPAEC. HPVEC overexpress development-associated genes (gremlin, mesenchyme homeobox 2, stem cell protein DSC54) and show an enhanced differentiation potential into adipocytes and osteoblasts in contrast to HPAEC. These data provide collective evidence for a juvenile venous and a more mature arterial phenotype of human fetal endothelial cells. The high plasticity of the fetal venous endothelial cells may reflect their role as tissue-resident endothelial progenitors during embryonic development with a possible benefit for regenerative cell therapy.

Insulin control of placental gene expression shifts from mother to foetus over the course of pregnancy

Aims/hypothesisThe human placenta is a complex organ situated at the interface between mother and foetus that separates maternal from foetal blood. The placental surfaces exposed to the two bloodstreams are different, i.e. trophoblasts and endothelial cells are in contact with the maternal and foetal circulation, respectively. Both cell types produce high insulin receptor levels. The aim of the present study was to test the hypothesis that spatio-temporal changes in insulin receptor expression in trophoblasts from first trimester to the endothelium at term shift the control of insulin-dependent processes from mother to foetus.MethodsGlobal microarray analysis of primary trophoblasts from first trimester and term human placentas and endothelial cells from term human placentas cultured under hyperinsulinaemic and control conditions identified different sets of regulated genes in trophoblasts and endothelial cells.ResultsInsulin effects on placental gene expression underwent developmental changes from trophoblasts in the first trimester to endothelial cells at term that were paralleled by changes in levels of activated insulin receptors. The changes in gene regulation were both quantitative (i.e. magnitude of effect) and qualitative (i.e. specific genes affected and direction of regulation).Conclusions/interpretationThis spatio-temporal shift in insulin sensitivity throughout pregnancy allows maternal and foetal insulin to regulate different processes within the placenta at different gestational stages, facilitated by compartmentalisation of the insulin response. Thus, by altering the levels and function of insulin receptors in space and time, control of insulin-dependent processes in the human placenta will change from mother to foetus throughout gestation. This will be of particular interest in conditions associated with altered maternal or foetal insulin levels, i.e. diabetes mellitus or intrauterine growth restriction.