Mechanotransduction at the cell-matrix interface.

The ability of cells to sense and respond to mechanical signals is vital in development and healthy tissue functioning. Many diseases are related to either changing mechanical properties of the tissue, or changes in the ability of cells to sense mechanical signals. This sensing occurs, in part, at integrin-associated complexes (IACs) that form sites of attachment between the cell and the extracellular matrix (ECM). In this review, we discuss the complex mechanical signals of the ECM. We will also outline how IACs are involved in cellular sensing of these mechanical properties, focussing on the molecular mechanisms of key adhesion molecules. Finally, the cellular mechanisms of mechanotransduction considering mechanosensing and signalling aspects of the core proteins in FAs are discussed and open questions outlined.

Contribution to the epidemiology of postnatal depression in Germany--implications for the utilization of treatment.

Using a longitudinal screening model, 772 mothers were screened for postnatal depression after delivery in Stuttgart (Germany). This model contained the Edinburgh Postnatal Depression Scale (EPDS) and the Hamilton Depression Scale (HAMD). The first screening was 6-8 weeks after delivery with the EPDS. Mothers with high scores in the first screening had a second screening 9-12 weeks after delivery with the EPDS at least three weeks after the first. Mothers with high scores in both screenings were investigated with the Hamilton Depression Scale (HAMD). Classification was performed with the DSM-IV. After observation until the third month after delivery, 3.6% (N = 28) of the 772 mothers were diagnosed with postnatal depression. Various methods of therapy were offered to those mothers. 18% (N = 5) accepted one or more of these methods of treatment. The rest of the mothers with postnatal depression refused--mostly for attitudinal or practical reasons. 13.4% of the mothers showed high scores in the first screening but not in the second. For those mothers a longitudinal observation is currently being performed to distinguish between a depressive episode and a depression with oscillating symptoms.

JAM-2, a novel immunoglobulin superfamily molecule, expressed by endothelial and lymphatic cells.

Cell-cell contacts are essential for morphogenesis and tissue function and play a vital role in mediating endothelial cohesion within the vascular system during vessel growth and organization. We identified a novel junctional adhesion molecule, named JAM-2, by a selective RNA display method, which allowed identification of transcripts encoding immunoglobulin superfamily molecules regulated during coculture of endothelial cells with tumor cells. The JAM-2 transcript is highly expressed during embryogenesis and is detected in lymph node and Peyer's patches RNA of adult mice. Accordingly, antibodies specific for JAM-2 stain high endothelial venules and lymphatic vessels in lymphoid organs, and vascular structures in the kidney. Using real time video microscopy, we show that JAM-2 is localized within minutes to the newly formed cell-cell contact. The role of the protein in the sealing of cell-cell contact is further suggested by the reduced paracellular permeability of cell monolayer transfected with JAM-2 cDNA, and by the localization of JAM-2 to tight junctional complexes of polarized cells. Taken together, our results suggest that JAM-2 is a novel vascular molecule, which participates in interendothelial junctional complexes.

Marching at the front and dragging behind: differential alphaVbeta3-integrin turnover regulates focal adhesion behavior.

Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain. At the cell front, adhesion sites containing alphaVbeta3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration.

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein-transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.

PECAM-1/CD31 trans-homophilic binding at the intercellular junctions is independent of its cytoplasmic domain; evidence for heterophilic interaction with integrin alphavbeta3 in Cis.

PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin alphavbeta3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin alphavbeta3 associates with PECAM-1 on the same cell surface as in a cis manner.

Actin dynamics in living mammalian cells.

The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human &bgr ;-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured 'actin clouds' were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

An epitope on VLA-6 (alpha6beta1) integrin involved in migration but not adhesion is required for extravasation of murine melanoma B16F1 cells in liver.

VLA-6 (alpha6beta1) integrin represents the major receptor for interaction with laminin substrate. It has been proposed that VLA-6 mediates tumor cell adhesion to the endothelium during extravasation. We have further explored this possibility using mouse melanoma B16F1 cells, which express VLA-6 as the principal laminin receptor, and two VLA-6 monoclonal antibodies (mAbs), MA6 and GoH3. Adhesion is a prerequisite of cell movement on matrix proteins. Thus, GoH3, which inhibited VLA-6-mediated adhesion, blocked cell movement on laminin. The recently prepared alpha6 integrin-specific mAb MA6 bound to an epitope in close proximity to GoH3, but it had no effect on VLA-6-mediated cell adhesion. We report here that although MA6 did not affect adhesion, it blocked mouse melanoma B16F1 cell movement on laminin to the same extent as GoH3. Results therefore demonstrate an active role of VLA-6 in providing cell movement as well as the initial adhesive event on laminin. In addition, mAb MA6 had no effect on the induction of tyrosine phosphorylation of focal adhesion kinase upon adhesion of B16F1 cells to laminin. Therefore, inhibition of cell movement by MA6 involved mechanism(s) other than an interference of VLA-6 signaling events leading to phosphorylation of focal adhesion kinase. The epitopes of GoH3 and MA6 may represent spatially and temporally related sites on VLA-6 that are involved during cell movement, or, alternatively, MA6 may inhibit the interaction of VLA-6 with associated cell surface molecules required for cell movement. In vivo videomicroscopy experiments also revealed that an inhibition of VLA-6 migratory function by MA6 resulted in a reduction in the ability of B16F1 to extravasate during hematogenous metastasis in the liver.

Integrin VLA-6 (alpha 6 beta 1) mediates adhesion of mouse bone marrow-derived mast cells to laminin.

The development of mast cells from bone marrow precursors and their function as the mucosal- or connective-tissue-type mast cell are critically dependent on microenvironmental factors. Extracellular matrix proteins, such as collagen, fibronectin, and laminin, may represent insoluble components of the microenvironment. Recent studies have described multiple isoforms of laminin isolated from different tissues. In the present study, adhesion of mouse bone marrow-derived mast cells (BMMC) and long-term mast cell lines to Engelbreth-Holm-Swarm (EHS) tumor laminin, rat laminin, human merosin, and human placental laminin was compared. The greatest level of adhesion was found with human laminin as the substrate. By use of a newly prepared mouse VLA-alpha 6 integrin-specific mAb (MA6) together with the previously described mAb GoH3, VLA-6 (alpha 6 beta 1) integrin was found to be expressed and utilized by BMMC and long-term mast cell lines. VLA-6 has been described as a major laminin receptor with roles in diverse cell functions including cell growth and differentiation. BMMC have been shown to express a 32/67-kDa laminin receptor. Therefore, in addition to the 32/67-kDa laminin receptor described in early studies, BMMC also express VLA-6 integrin, which may have roles in the regulation of their development.

Differential utilization of VLA-4 (alpha 4 beta 1) and -5 (alpha 5 beta 1) integrins during the development of mouse bone marrow-derived mast cells.

Cytokines have been shown to have major roles in the development of mast cells from bone marrow progenitors. Immature mast cells derived from bone marrow thus leave the blood system to complete their course of maturation within tissues. However, it is now clear that VLA (beta 1) integrins with function in mediating cell-cell and cell-extracellular matrix protein interactions have effects on the growth and differentiation of diverse cell types. At present, the involvement of VLA integrins during mast cell development is still unclear. In this study, we report the preparation of a new monoclonal antibody (mAb) against mouse VLA-5 (alpha 5 beta 1) integrin. Together with mAb R1-2, we characterized the expression of VLA-4 (alpha 4 beta 1) and VLA-5 integrins, the two major fibronectin receptors, on two long-term cultured mast cell lines, CFTL-15 and MC/9. CFTL-15 cells were found to express both VLA-4 and -5 integrins whereas MC/9 cells expressed only VLA-5 but not VLA-4. We speculated that VLA integrin expression may be related to mast cell development. Thus bone marrow-derived mast cells (BMMC) were characterized after varying periods of development induced by IL-3. During the first 3 weeks the expression of VLA-4 and VLA-5 increased progressively and both were involved in mediating adhesion of BMMC to fibronectin. At time periods of greater than 3 weeks, the expression of VLA-4 declined gradually to little, if any, by week 13. In comparison, VLA-5 remained stably expressed and functioned as the major receptor for fibronectin. Results from this study therefore suggest that BMMC differentially utilize VLA-4 and VLA-5 integrins during IL-3-induced development. Differential expression of VLA integrins may have effects on the recirculation properties, tissue distribution and eventual maturation of progenitors to fully matured mast cells.

Integrin VLA-2 (alpha2beta1) function in postextravasation movement of human rhabdomyosarcoma RD cells in the liver.

It is now known that members of the selectin and integrin families are critical in the initial interaction of cells in circulation with endothelial surfaces. Also, platelet/endothelial cell adhesion molecule-1 has been shown to be involved in transendothelial migration of extravasating cells. Little is known about adhesion molecules involved in subsequent postextravasation events. In this study, the significance of VLA-2 (alpha2beta1) integrin in the movement of human rhabdomyosarcoma RD cells in the liver was characterized by in vivo videomicroscopy. Results show that after extravasation, the mock-transfected RDpF cells were able to migrate to the subcapsular region of the liver. Although the RDX2C2 transfectant expressing VLA-2 integrin extravasated equally well, a majority of RDX2C2 cells remained in close proximity to blood vessels and failed to reach the subcapsular region. The functional involvement of VLA-2 in affecting the ability of RD cells to reach the subcapsular region was verified by the preparation of an RD transfectant [RDX2C2(I-)] expressing a nonfunctional variant of VLA-2 lacking the inserted (I)-domain of alpha2 subunit. In vivo microscopy showed that RDX2C2(I-) cells migrated in a manner similar to control RDpF cells. To demonstrate that RDX2C2 cells that remained in dose proximity to blood vessels were due to VLA-2 function, a blocking monoclonal antibody against VLA-2 (BHA2.1) was prepared. Mice were injected with BHA2.1 or control monoclonal antibody P3 at the time when RDX2C2 cells completed their extravasation. Treatment with BHA2.1 increased the number of RDX2C2 cells that reached the subcapsular region and subsequently formed tumor foci. Therefore, VLA-2 integrin expression has major roles in postextravasation movement and affects tumor foci formation at the liver surface.

VLA-beta 1 integrin subunit-specific monoclonal antibodies MB1.1 and MB1.2: binding to epitopes not dependent on thymocyte development or regulated by phorbol ester and divalent cations.

We report here the isolation of two new monoclonal antibodies (MB1.1 and MB1.2) against mouse VLA-beta 1 integrin subunit. Characterization by flow cytometry demonstrated binding of MB1.1 and MB1.2 to freshly isolated thymocytes, primary bone marrow mast cell lines, as well as cell lines of distinct lineage each expressing different combination of VLA integrins. The specificity of MB1.1 and MB1.2 was determined by (1) their binding to antigen with M(r) about 120 kDa, and (2) the ability of antiserum against the carboxyl terminal of VLA-beta 1 subunit to deplete antigens for MB1.1 and MB1.2 in sequential immunoprecipitation experiments. The epitopes for MB1.1 and MB1.2 were in close proximity to each other since preincubation of cells with one MAb inhibited the binding of the other. However, MB1.1 and MB1.2 differed in their affinity for the beta 1 subunit. In addition, neither MAbs had any effect on cell adhesion to matrix proteins indicating that the epitopes involved are distant from VLA integrin ligand-binding sites. MB1.1 and MB1.2 appear to differ from the two MAbs so far reported against mouse VLA-beta 1 subunit, KMI6 and 9EG7. Thus, the epitopes for MB1.1 and MB1.2 were readily detectable on unfractionated thymocytes whereas KMI6 has been reported to bind only a fraction of CD4-8- and CD4-8+ thymocytes. Phorbol ester and Mn2+, which have been shown to regulate the binding of 9EG7, had no effect on MB1.1 and MB1.2 binding to VLA-beta 1 integrin subunit.

Jumping translocation in a phenotypically normal female.

"Jumping translocation" jt refers to a rare type of chromosome mosaic, in which the same portion of a (donor) chromosome is translocated to different (recipient) chromosome sites. Jt have mainly been observed in lymphocyte cultures of patients with hematologic malignancies. We report a phenotypically normal female carrying a mosaic of two cell lines with the Xq26-qter segment translocated to the short arm of chromosomes 15 or 21 in peripheral blood lymphocytes. In skin fibroblasts, only the X/21 translocation was detected. We speculate that recombination between homologous repetitive sequences on non-homologous human acrocentrics may be the cause of such chromosomal rearrangements.

Rearrangement of chromosome 1 is a frequent finding in endometrial carcinoma. An in situ hybridization study in nine endometrial carcinomas.

Nine endometrial carcinomas were examined for numerical aberrations of the chromosomes 1,7, and X by fluorescence in situ hybridization using highly repetitive chromosome-specific probes. In addition, a combination of a centromeric and a telomeric chromosome 1 probe was applied to detect structural chromosome 1 aberrations. Chromosome aberrations were found in six tumors. In four of these, an imbalance between 1q12 and 1p36 was detected, indicating the presence of an extra 1p- chromosome. In regard to the chromosomes 7 and X, monosomies and trisomies were found. Intratumoral genetic heterogeneity in endometrial carcinomas was detectable by FISH and flow cytometry. In conclusion, our findings confirm that chromosome 1 is frequently involved in structural chromosome changes, indicating chromosome 1 to be of importance in the evolution of endometrial carcinoma.

[Correlation of cotinine level in amniotic fluid, umbilical artery blood and maternal blood]. / Korrelation des Cotininspiegels in Fruchtwasser, Nabelarterienblut und mütterlichem Blut.

The aim of the present study was to determine the correlation of cotinine levels in amniotic fluid and in fetal and corresponding maternal blood samples. Amniotic fluid samples (N = 130) were taken during second trimester amniocentesis, umbilical artery blood samples (N = 75) at birth, both together with corresponding maternal blood samples. Self-reported smokers showed maternal serum cotinine levels > 15 ng/ml in 93%, self-reported nonsmokers levels < 15 ng/ml in 89%. Correlation of corresponding values for cotinine was 0.81-0.92. Cotinine values were increased in fetal blood and amniotic fluid in comparison to maternal serum levels. Despite the fact that pathophysiology is not fully understood, an accumulation of nicotine and its metabolites both in the fetus and in the amniotic fluid appears to be evident.

[Antepartum monitoring and peripartal findings in mono- and dichorial twin pregnancy]. / Antepartale Uberwachung und peripartale Befunde bei mono- und dichorialer Zwillingsschwangerschaft.

In a group of 65 twin pregnancies the difference of perinatal findings and antepartal test results was evaluated in relation to amnionicity and chorionicity. Monochorionic placentation was found in 33% of the pregnancies. The rate of foetal malformation (11%), neuromuscular dysfunction (6%), perinatal mortality (11%) and duration of neonatal intensive care was increased in those cases. The most useful diagnostic tool was B-Mode-ultrasound (first detection and surveillance of multiple pregnancy, especially diagnosis of inter-twin growth discordancy). Non stress test and Doppler sonography were found to be of value as additional tests for detection of functional differences between both twins. There were no differences between findings in first and second twin as well as between findings in pregnancies with mono- or dichorionic placentation.

[Premature separation of the placenta--clinical findings and fetal monitoring]. / Vorzeitige Plazentalösung--klinische Befunde und fetale Uberwachungsmethoden.

In a study group of 41 pregnant women of postpartally confirmed placental abruption, the prognostic value of clinical and diagnostic findings was investigated. The incidence of placental abruption was 1.4% of all deliveries within a three-year interval. 51% of patients showed vaginal bleeding before delivery. Retroplacental haematoma was found in 49% of cases ultrasonographically. A total of 75% had a pathological CTG test. More than 95% of these findings occurred within 3 days before delivery. Abnormal Doppler flow findings in foetal vessels more than 3 days before delivery were seen in 62% of cases. In the last three days before delivery, 86% were abnormal. Preterm delivery before 37 weeks of gestation was registered in 82% of patients. Perinatal mortality amounted to 12%. The rate of severely dystrophic newborn was 30%. Even in cases with lack of the clinical and/or sonographical findings, the possibility of placental abruption should be considered, if an acute deterioration of cardiotocographic or Doppler-sonographic findings.

[Evaluation of the antepartum CTG course in cases of highly pathologic Doppler flow findings]. / Auswertung des antepartalen CTG-Verlaufs in Fällen mit hochpathologischen Doppler-Flow-Befunden.

In a group of 79 pregnancies with highly abnormal Doppler-flow findings in foetal vessels the value of antepartal CTG criteria was evaluated in detail. In cases with absent enddiastolic flow (AEDF) the prognostical value was not further increased by antepartal pathological CTG findings. In cases with permanently normal antepartal CTG's, however, foetal outcome was fairly good (median duration of pregnancy 38 + 6 weeks, median foetal birth weight 2,545 grams, arterial pH < 7.20 in 9%). This was true also in the presence of pathological Doppler-flow findings except AEDF. In cases with pathological antepartal CTG findings in the antepartal course of pregnancy the loss of acceleration was most frequent (82%), followed by decreased frequency (53%) or narrowed amplitude (38%) of the foetal heart rate variation. Combination of both methods (Doppler sonography and CTG) is recommended clinically because the rate of uncertain findings can be reduced. Especially in cases with AEDF an active management would be justified before CTG's become pathological.

[Doppler ultrasound of fetal blood vessels--optimizing the diagnostic value by a combined diagram]. / Doppler-Sonographie der fetalen Gefässe--Optimierung der Aussagekraft durch ein Kombinationsdiagramm.

The dopplersonographic data of 1926 pregnant women in the 3. trimester of pregnancy was used to evaluate a graphic system for the analysis of A/B-ratio in fetal vessels (pulsed wave duplex system; descending aorta and umbilical arteries). The basis of the newly developed combination diagram were dopplersonographic standard values (single cut off and pregnancy duration related percentiles). One of the results was the fact, that the combined measurement and analysis of both fetal vessels increased the positive predictive value compared to single measurements. The value of dopplersonographic examinations in the aorta was slightly better than those in the umbilical arteries. The combined diagram showed a marked improvement in graphicness, especially in follow-up cases, and a partial improvement in statistical values.