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Biomedical subjects

H G Frank

Publications and source records attributed to H G Frank.

At least 19 recordsLinked to original sources
PubMed2001

Macrophage-induced apoptosis limits endovascular trophoblast invasion in the uterine wall of preeclamptic women.

Impaired invasion of uteroplacental arteries by extravillous trophoblast cells is a key pathogenic mechanism of preeclampsia. We previously demonstrated that reduced trophoblast invasion into uteroplacental spiral arteries was associated with an excess of macrophages in and around these arteries. To explore the significance of these observations, we correlated the extent of extravillous trophoblast apoptosis in placental bed biopsy specimens with macrophage distribution and studied the effect of macrophages upon trophoblast apoptosis in vitro. Extravillous trophoblast hybrid cells were cocultured with activated macrophages exposed to exogenous tumor necrosis factor alpha (TNFalpha), anti-tumor necrosis factor receptor I (TNF-RI), and tryptophan depletion, and the rates of trophoblast apoptosis were measured. Extravillous trophoblast hybrid cells showed increased rates of apoptosis following exposure to exogenous TNFalpha, with tryptophan depletion, and when cocultured with activated macrophages. The proapoptotic effects of macrophages in vitro were completely inhibited only by simultaneous addition of tryptophan and anti-TNF-RI. Our data indicate that macrophages, residing in excess in the placental bed of preeclamptic women, are able to limit extravillous trophoblast invasion of spiral arterial segments through apoptosis mediated by the combination of TNFalpha secretion and tryptophan depletion. The mechanisms by which macrophages are activated and recruited to the placental bed are presently unknown but are likely central to the pathogenesis of preeclampsia.

Adult↗
PubMed2001

Human trophoblast contains an intracellular protein reactive with an antibody against CD133--a novel marker for trophoblast.

CD133 is a protein expressed on the cell membrane of a subfraction of haematopoietic stem and progenitor cells, as well as on some epithelial cells. Previously available antibodies against CD133 recognized only the glycosylated protein, localized to membrane protrusions or microvilli. Due to this, immature intracellular stages of the CD133 protein could not be visualized using these antibodies. We describe reactivity of a commercially available antibody against CD133, called AC133-2, with an intracellular protein in trophoblast. Both villous and extravillous cytotrophoblast, as well as syncytiotrophoblast were stained by AC133-2 in cryostat sections of first trimester and term placenta. Villous stroma was not stained. AC133-2 reactivity was seen in methanol-fixed primary trophoblast cells and trophoblast-derived cell lines, and was coexpressed with cytokeratin-7. CD133 messenger RNA was present in trophoblast and trophoblast-derived cell lines, but also in cells not displaying any reactivity with CD133 antibodies. AC133-2 recognized a 55-60 kDa protein on Western blots of cell extracts including trophoblast. The exact nature of this protein is not yet understood. However, AC133-2 is applicable as a positive marker for the characterization of all subtypes of trophoblast and for trophoblast cell lines.

AC133 Antigen↗
PubMed2000

Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid.

We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.

Cell Fusion↗
PubMed2000

Expression of the imprinted genes MEST/Mest in human and murine placenta suggests a role in angiogenesis.

In the mouse fetus, Mest is widely expressed in mesoderm derived tissues. In separate studies in mice and in humans, it has been shown to be maternally imprinted, that is, only the paternally inherited allele is active. Here, we show that starting with implantation, Mest is also expressed in maternal decidua of the mouse and in placenta of both humans and mice. Expression in murine decidua was restricted to endothelial cells. After Day 7, expression in the decidua gradually decreased. Mest-specific RT-PCR and restriction fragment length variant (RFLV) analysis of decidualized endometrium isolated from (M. musculus x M. spretus)F1 females showed that only the paternally derived Mest allele was activated in the decidual endothelium. In the mouse extraembryonic tissues, Mest transcripts were detected in derivatives of extraembryonic mesoderm only. Here, hemangioblast precursor cells and endothelial cells were positive. At all developmental stages of the mouse, trophoblast-derived cells were clearly devoid of Mest transcripts. In the human placenta MEST transcripts were also detected in hemangioblast precursor cells, however, MEST was also expressed in villous and invasive cytotrophoblast. In a human choriocarcinoma/trophoblastic tumour grown in a nude mouse, human MEST was expressed in the tumour cells, whereas murine Mest was expressed in endothelia of the murine capillaries. The expression pattern exhibited by both Mest and MEST in extraembryonic tissues during development and during formation of choriocarcinoma/trophoblast tumour suggests a functional role of the MEST proteins related to oncofetal angiogenesis. Dev Dyn 2000;217:1-10.

Animals↗
PubMed2000

The human placenta is encircled by a ring of smooth muscle cells.

The marginal zone of the human term placenta was studied by transmission electron microscopy and immunohistochemistry using antibodies against cytoskeletal filaments, extracellular matrix molecules and endothelial markers. The marginal sinus of the intervillous space is separated from the chorionic and basal plates by a layer of cells expressing vimentin, desmin, alpha- and gamma-smooth muscle actins, and smooth muscle myosin. Also ultrastructurally, these cells share all features with smooth muscle cells. This muscular ring is continuous with the media of uteroplacental veins entering the marginal sinus. In the basal plate the muscle cells may extend far into the central parts of the placenta. The muscular ring is separated from the intervillous space by a layer of endothelial cells. They are continuous with the maternal endothelium of the marginal uteroplacental veins. Moreover this endothelium covers neighbouring parts of the chorionic and basal plates, locally extending to the surfaces of large stem villi. The data suggest (1) that the marginal zone of the intervillous space ('marginal sinus') represents the dilated and merged parts of uteroplacental veins and (2) that lateral growth of the human placenta partly takes place by expansion into the uteroplacental veins. The functional importance of this muscular ring remains unknown.

Actins↗
PubMed2000

Pregnancy-induced de-differentiation of media smooth muscle cells in uteroplacental arteries of the guinea pig is reversible after delivery.

The majority media of smooth muscle cells of uteroplacental arteries of the guinea pig is not destroyed during trophoblast invasion. Rather, most of these cells de-differentiate during pregnancy-induced arterial dilatation, forming a population of mesenchyme-like myoblasts ready to reconstitute the media after birth. We have studied the re-differentiation of these cells after delivery by means of transmission electron microscopy and immunohistochemistry using antibodies against a panel of cytoskeletal proteins. The data reveal that post partum re-differentiation of the media myoblasts starts immediately after birth where some of the invasive trophoblast cells are still present. The process of re-differentiation is completed at day 8 after parturition. Post partum re-differentiation can be subdivided into two steps: until day 5 after parturition, the central parts of the media are reconstituted out of the reservoir of vimentin-positive myoblasts by stepwise acquisition of desmin, alpha-smooth muscle actin, gamma-smooth muscle actin and smooth muscle myosin. Only thereafter the same re-differentiation takes place in the peripheral parts of the media. On the ultrastructural level immunohistochemical re-expression of cytoskeletal proteins is accompanied by reconstitution of the intracellular contractile apparatus. The data support our earlier notion that the majority of media smooth muscle cells in the guinea pig uterus does not degenerate during trophoblast-invasion but rather de-differentiate temporarily.

Animals↗
PubMed1999

Developmental expression patterns of connexin26 and -30 in the rat cochlea.

Connexin proteins form transmembranous gap junction channels that connect adjacent cells. Connexin26 and connexin30 have been previously shown to be strongly expressed in the inner ear of adult rats and to be mainly colocalized. Because intercellular connections by gap junction proteins are crucial for maturation of different tissues, we investigated the developmental expression of connexin26 and connexin30 in pre- and postnatal rats using immunocytochemistry. In the rat otocyst, staining for connexin26 as well as for connexin30 appeared at the 17th day of gestation. However, at this stage, expression of connexin30 was low and restricted to the neurosensory epithelium. Beginning from the 3rd postnatal day connexin26 and -30 were expressed with highest immunoreaction in the spiral limbus, the neurosensory epithelium, and between the stria vascularis and the spiral ligament. Beginning from postnatal day 12 the staining pattern resembled that of adult animals, with additional strong staining between all fibrocytes of the spiral ligament. Double labeling experiments demonstrated strongest colocalization of both connexins between the stria vascularis and the spiral ligament. These results demonstrate that development of the cochlear gap junction system precedes the functional maturation of the rat inner ear, which takes place between the 2nd and 3rd postnatal week. In the cochlea of a 22-week-old human embryo, connexin26 and connexin30 could be detected in the lateral wall, suggesting that both connexins also play a crucial role in function of the human inner ear.

Animals↗
PubMed1999

The apoptosis cascade--morphological and immunohistochemical methods for its visualization.

Apoptosis is involved in morphogenesis of embryonic tissues as well as in homeostasis of adult organs and tissues. It is the main process by which organs maintain cell mass and at the same time eliminate excess and aged cells that have lost their functional importance. The typical morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events, some of which are inextricably linked to the process of differentiation. Studies that analyze all stages of this cascade, rather than the final morphological stages of apoptotic death, are essential in order that specific link(s) between differentiation and apoptosis are appreciated. This review outlines the main stages of the apoptosis cascade together with current methods for their morphological visualization. Starting with (a) receptors and ligands known to induce apoptosis, we continue with (b) early initiator stages of apoptosis, and (c) proteins regulating and potentially inhibiting further progression of the cascade, into (d) irreversible execution stages of the cascade, and finally (d) the morphological events of apoptotic death. For each stage we present those aspects of the biochemical background that are morphologically relevant, together with proven methods for their visualization. We offer technical advice at each stage based upon our experience of studying differentiation and apoptosis in human placental trophoblast.

Animals↗
PubMed1999

Apoptosis cascade progresses during turnover of human trophoblast: analysis of villous cytotrophoblast and syncytial fragments in vitro.

Growth and survival of the trophoblast layer of the human placenta depends on continuous incorporation of villous trophoblast stem cells (cytotrophoblast), by syncytial fusion, into the syncytiotrophoblast. Descriptive studies suggest that this process may be intimately related to apoptosis. We have analyzed the expression and activation of initiator and execution caspases, critical effectors of apoptosis, in relation to trophoblast turnover (differentiation) in human placental villi. We used immunohistochemistry, caspase enzyme histochemistry, caspase activity assays, Western blots, and autoradiography techniques on placental tissue sections, trophoblast lysates, villous explants, and isolated trophoblast fragments (villous cytotrophoblast and mononuclear syncytial elements) in vitro. Our data demonstrate expression of initiator caspases 8 and 10 and activity of caspase 8 in villous cytotrophoblast. Proforms of the execution caspases 3, 6, and 7 were also expressed in villous cytotrophoblast, but activation of execution caspases 3 and 6 could only be demonstrated in the syncytiotrophoblast after syncytial fusion. Down-regulation of the general transcription level (reduced incorporation of [3H]uridine) as well as cleavage of the execution caspase substrates poly-(ADP-ribose)polymerase and lamin B was confined to syncytiotrophoblast and preceded the final events of apoptotic death (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling reactivity and nuclear collapse). Our data confirm that the apoptosis cascade in villous trophoblast is regulated in parallel with trophoblast differentiation, syncytial fusion, and trophoblast turnover.

Apoptosis↗
PubMed1998

Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta.

Villous trophoblast in the human placenta consists of a population of proliferating stem cells which differentiate and individually fuse into the syncytiotrophoblast. We studied the apoptotic cascade in this complex epithelial layer by immunohistochemical localization of Fas, FasL, Bcl-2, Mcl-1, pro-caspase-3 and caspase-3, T-cell-restricted intracellular antigen-related protein (TIAR), poly(ADP-ribose) polymerase (PARP), lamin B, topoisomerase IIalpha, and transglutaminase II in cryostat and paraffin-fixed tissue sections from normal human first-trimester and term placental villi. The relationship between the apoptotic cascade and syncytial fusion was studied by coincubation of intact villi with FITC-coupled annexin-V, to detect the phosphatidylserine flip, and propidium iodide, to detect plasma membrane permeability. The final events of the apoptotic cascade were studied by the TUNEL reaction and ultrastructural appearance of the trophoblast. The phosphatidylserine flip was identified in some of the villous cytotrophoblastic cells, but the presence of both Bcl-2 and Mcl-1 proteins presumably prevented continuation of the apoptotic cascade. The syncytiotrophoblast demonstrated heterogeneous findings, suggesting variable progression along the apoptotic cascade. In some areas Bcl-2 and Mcl-1 predominated, with preservation of the nuclear proteins PARP, lamin B, and topoisomerase IIalpha; in other areas, especially in and around syncytial sprouts, Bcl-2 and Mcl-1 were absent, accompanied by loss of nuclear proteins, presence of phosphatidylserine flip, and TUNEL positivity. These data suggest that the apoptotic cascade is initiated in the villous cytotrophoblast, which in turn promotes syncytial fusion. Donation of anti-apoptotic proteins into the syncytium, such as Bcl-2 and Mcl-1, focally inhibits further progression along this cascade. Completion of the apoptotic cascade takes place in and around syncytial sprouts, providing further evidence that these are the sites of trophoblast shedding into the maternal circulation.

Apoptosis↗
PubMed1998

Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta.

The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.

Collagen↗
PubMed1997

Transport and fate of trifluoroacetate in upland forest and wetland ecosystems.

Although trifluoroacetate (TFA), a breakdown product of chlorofluorocarbon replacements, is being dispersed widely within the biosphere, its ecological fate is largely unknown. TFA was added experimentally to an upland, northern hardwood forest and to a small forest wetland ecosystem within the Hubbard Brook Experimental Forest in New Hampshire. Inputs of TFA were not transported conservatively through these ecosystems; instead, significant amounts of TFA were retained within the vegetation and soil compartments. More TFA was retained by the wetland ecosystem than by the upland forest ecosystem. Using simulation modeling, TFA concentrations were predicted for soil and drainage water until the year 2040.

Adsorption↗
PubMed1996

Extracellular matrix components of the placental extravillous trophoblast: immunocytochemistry and ultrastructural distribution.

Invasive extravillous trophoblast cells of the human placenta are embedded in a self-secreted extracellular matrix, the matrix-type fibrinoid. The ultrastructure and molecular composition of the matrix-type fibrinoid of the term human placenta were studied by transmission electron microscopy and immunogold labelling. We used antibodies directed against different matrix proteins such as collagen type IV, laminin, vitronectin, heparan sulfate, various fibronectin isoforms, and against the oncofetal blood group antigen, "i". Immunogold labelling patterns of matrix proteins are the basis for the subdivision of the trophoblast-derived matrix-type fibrinoid into mosaic-like patches of structurally and immunocytochemically different compartments. Firstly, fine granular patches with structural similarities to basal lamina material are composed solely of collagen type IV and laminin. Secondly, an ultrastructurally amorphous glossy substance shows reactivity with antibodies against heparan sulfate and vitronectin. A third type of patches, fine fibrillar networks embedded in the above-mentioned glossy matrix, are reactive with antibodies against normal fibronectin isoforms (IST-4, IST-6, IST-9) and oncofetal isoforms (BC-1, FDC-6). The blood group precursor antigen "i" was not only expressed on the surfaces of the extravillous trophoblast cells but was associated with the fibronectin-positive fibrils. In conclusion, within this extracellular matrix, clear compartments of different composition can be distinguished from each other. Glycosylation with "i" in this matrix may be involved in immunological masking, thus preventing rejection of placenta and fetus.

Antibodies↗
PubMed1996

The fibrinoids of the human placenta: origin, composition and functional relevance.

Placental fibrinoids are extracellularly deposited materials which are histologically glossy and acid staining, and can be found in every normal and pathological placenta at all stages of pregnancy. The amount of fibrinoid is, in general, independent of pregnancy outcome and fetal wellbeing. According to new findings, the classical histological term "fibrinoid" covers two distinctive extracellular matrices which differ as regards structure, composition and function. Fibrin-type fibrinoid is mostly composed of fibrin together with other molecules derived from blood clotting or degenerative processes. It is mainly a maternal blood-clot product which is used (a) to adapt the intervillous space to optimized flow conditions and (b) to control growth of the villous trees by encasing new villous branches which caused intervillous stasis or turbulence of maternal blood. Moreover, fibrin-type fibrinoid replaces degenerative syncytiotrophoblast at the maternofetal exchange surfaces, thus acting as a kind of substitute barrier. Matrix-type fibrinoid is a secretory product of invasive extravillous trophoblast cells. It shares some similarities with basement membranes, however, it is secreted in an apolar fashion, embedding the secreting cells. Like basement membranes, it contains laminins, collagen IV, and heparan sulfate. In addition, oncofetal fibronectins, vitronectin, and i-glycosylated molecules but no collagens I, III, and VII can be found. Matrix-type fibrinoid is thought to regulate trophoblast invasion by specific interactions with cell surface integrins. As a kind of "glue", it anchors the placenta to the uterine wall and seems to play an important role in materno-fetal immune interactions at this particular site. Both types of fibrinoid are usually co-localized, thus indicating close morphogenetic and functional interrelations.

Animals↗
PubMed1995

Physiological dilation of uteroplacental arteries in the guinea pig depends on nitric oxide synthase activity of extravillous trophoblast.

The trophoblast invasion of uteroplacental arteries in the guinea pig has been studied by means of electron microscopy and immunohistochemisty. To identify trophoblast cells, smooth muscle cells, and endothelial cells, antibodies against cytokeratins, smooth muscle myosin, desmin, and vimentin were employed. Furthermore, the immunohistochemical expression patterns of nitric oxide synthase isoforms (eNOS, mNOS and bNOS) were studied and were compared with the enzyme histochemical staining for NADPH-diaphorase. Dilation of uteroplacental arteries begins prior to day 30, when trophoblast cells that coexpress endothelial and macrophage nitric oxide synthase can be found in the vicinity of the vessels and replace the surrounding peritoneal mesothelium. Trophoblast invasion of the arterial walls and the subsequent wall destruction are only secondary effects. Starting around day 50, the final steps of pregnancy-dependent vessel modifications involve intraarterial trophoblast adhesion to the endothelium and subsequent replacement of the endothelium by the trophoblast cells. These may centrifugally invade the vessel media eventually forming intraluminal plugs. These findings led us to the conclusion that in the guinea pig pregnancy-induced physiological dilation of the uteroplacental arteries is due to the effect of nitric oxide rather than being caused by trophoblast-induced media destruction.

Animals↗