Abstract. Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an Mr-200 kD integrin oL subunit that associates with the integrin ~, subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the ot subunit of the human integrin VLA-1. The full-length rat oL1 cDNAs encode a protein containing a putative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin o~ chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg 2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat ¢x~ sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-l, LFA-1, p150). The rat o~t and human VLA-2 ¢x subunits share >50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin ¢x~ subunit has several unique features, including a 38-residue insert between two Ca2+/Mg 2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.

The complement system plays an important role in normal human pregnancy. Uncontrolled activation of this system has been associated with many disease states. We tested the hypothesis that the C5b-9 membrane attack complex (MAC) localizes to sites of villous injury and modulates trophoblast function. Placental sections from pregnancies with no complications, intrauterine growth restriction, or preeclampsia were immunostained and the surface density for MAC and fibrin was determined by morphometric analysis. Primary cytotrophoblasts from term placentas were cultured in a FiO2 of < 1%, 8% and 20% with 10% human serum containing active MAC or heat-inactivated control serum. Immunofluorescent MAC binding to trophoblast was quantified, and the neoepitopes formed in cytokeratin 18 filaments and poly-ADP-ribose polymerase during apoptosis were used to measure cell death. Trophoblast differentiation was assessed by HCG secretion, formation of syncytia, and expression of syncytin. MAC localized to fibrin deposits in normal placentas, and especially in placentas from IUGR and preeclampsia. MAC binding to cytotrophoblasts was inversely proportional to FiO2 and enhanced apoptosis. MAC increased markers of differentiation in cultures at 72h (medium HCG, syncytia and syncytin expression). Our findings demonstrate that MAC associates with fibrin deposits at sites of villous injury in vivo. Hypoxia also enhances MAC deposition in cultured trophoblasts and MAC alters trophoblast function in a phenotype specific manner.

Human placental villi are surfaced by the syncytiotrophoblast, a multinucleated, epithelial-cell layer that functions in maternal-fetal exchange. Mononucleated cytotrophoblasts are subjacent to the syncytiotrophoblast. Using confocal fluorescence microscopy of third-trimester villi, we previously found that cytotrophoblasts are often interdigitated into the syncytiotrophoblast, that cytotrophoblasts undergo caspase-mediated apoptosis, and that apoptosis is much lower, and perhaps completely inhibited, in intact syncytiotrophoblast lacking fibrin-type fibrinoid. Previous analysis of primary cultures of human trophoblasts also indicated lower levels of apoptosis in syncytiotrophoblast compared to cytotrophoblasts. Here, using confocal microscopy we find that cultured cytotrophoblasts and syncytiotrophoblasts display complex structural relationships, as in vivo, and that apoptosis of a cytotrophoblast or syncytiotrophoblast does not induce apoptosis of adjacent trophoblasts. Using live-cell imaging of mitochondrial depolarization and nuclear condensation in cultured syncytiotrophoblasts, we show apoptosis initiates in a localized region and propagates radially at ~five μm/min with no loss of velocity until the entire syncytium has undergone apoptosis. The rate of propagation is similar in cases of spontaneous apoptosis and in apoptosis that occurs in the presence of cobalt chloride or rotenone, two inducers of apoptosis. We suggest that inhibition of syncytiotrophoblast apoptosis in vivo is important to prevent widespread syncytiotrophoblast death, which would result in placental dysfunction and contribute to poor pregnancy outcomes.

We tested the hypothesis that epidermal growth factor (EGF) limits hypoxia-induced apoptosis in cultured human trophoblasts by phosphorylation of the proapoptotic protein Bcl-2-associated death promoter (BAD). Cytotrophoblasts were isolated from placentas of uncomplicated pregnancies at 38-40 wk gestation. Primary trophoblasts or transfected JEG3 trophoblast cells were cultured in less than 1 or 20% oxygen in the presence or absence of EGF and signaling pathway inhibitors. BAD, green fluorescent protein (GFP)-BAD, 14-3-3, Bcl-X(L), and neoepitopes formed during apoptotic cleavage of cytokeratin 18 intermediate filaments were quantified using immunoblotting. Cultures immunostained by fluorescent antibodies were analyzed by confocal microscopy for BAD and GFP. Fluorescence resonance energy transfer was used to detect molecular interaction between endogenous BAD and GFP-BAD. We found EGF increased the phosphorylation of BADser112 under standard culture conditions. Whereas hypoxia enhanced apoptosis and increased phosphorylation of both BADser136 and BADser155, hypoxia diminished phosphorylation of BADser112, and this effect was reversible by EGF. Transfected GFP-BAD, which directly interacted with endogenous BAD by colocalization and fluorescence resonance energy transfer, enhanced hypoxia-induced apoptosis in JEG3 cells. EGF reduced apoptosis in hypoxic JEG3 cells that overexpressed GFP-BAD but not in cells overexpressing GFP-BAD that harbored a serine-to-alanine mutation at the 112 site. Coimmunoprecipitation studies showed that EGF reduced the proapoptotic interaction of BAD with Bcl-X(L). The effect of EGF on phosphorylation of BADser112 was dependent on the action of p38 MAPK. We conclude that EGF signals via p38 MAPK to increase phosphorylation of BADser112 and thereby limit trophoblast apoptosis.

AIMTo investigate validity of electrocardiographic (ECG) criteria for left ventricular hypertrophy (LVH) in young adults.METHODSRetrospectively, echocardiograms showing LVH and concomitant electrocardiograms were collected in patients 18 to 39 years old. A control group of patients without LVH was collected. Using echocardiogram as the gold standard, electrocardiograms were analyzed using common voltage criteria.RESULTSStudy included 100 subjects (52% male, mean age = 28 ± 6.8 years, 96% Hispanic or African-American) with 50% LVH prevalence. Sensitivity and specificity for Sokolow-Lyon criteria were 24% (95%CI: 13.5%-38.4%) and 88% (95%CI: 74.9%-95%). For Cornell criteria, sensitivity was 32% (95%CI: 19.9%-46.8%) and specificity 98% (95%CI: 87.9%-99.8%). For R in aVL criteria, sensitivity was 12% (95%CI: 4.9%-25%) and specificity 100% (95%CI: 91.1%-100%).CONCLUSIONIn young adults common ECG voltage criteria have low sensitivities and high specificities similar to other age groups. Low sensitivities preclude these ECG criteria from serving as effective screening tests.