The long non-coding RNA NEAT1 is increased in IUGR placentas, leading to potential new hypotheses of IUGR origin/development.

INTRODUCTION: Intrauterine Growth Restriction (IUGR) is a multifactorial disease defined by an inability of the fetus to reach its growth potential. IUGR not only increases the risk of neonatal mortality/morbidity, but also the risk of metabolic syndrome during adulthood. Certain placental proteins have been shown to be implicated in IUGR development, such as proteins from the GH/IGF axis and angiogenesis/apoptosis processes. METHODS: Twelve patients with term IUGR pregnancy (birth weight < 10th percentile) and 12 CTRLs were included. mRNA was extracted from the fetal part of the placenta and submitted to a subtraction method (Clontech PCR-Select cDNA Subtraction). RESULTS: One candidate gene identified was the long non-coding RNA NEAT1 (nuclear paraspeckle assembly transcript 1). NEAT1 is the core component of a subnuclear structure called paraspeckle. This structure is responsible for the retention of hyperedited mRNAs in the nucleus. Overall, NEAT1 mRNA expression was 4.14 (±1.16)-fold increased in IUGR vs. CTRL placentas (P = 0.009). NEAT1 was exclusively localized in the nuclei of the villous trophoblasts and was expressed in more nuclei and with greater intensity in IUGR placentas than in CTRLs. PSPC1, one of the three main proteins of the paraspeckle, co-localized with NEAT1 in the villous trophoblasts. The expression of NEAT1_2 mRNA, the long isoform of NEAT1, was only modestly increased in IUGR vs. CTRL placentas. DISCUSSION/CONCLUSION: The increase in NEAT1 and its co-localization with PSPC1 suggests an increase in paraspeckles in IUGR villous trophoblasts. This could lead to an increased retention of important mRNAs in villous trophoblasts nuclei. Given that the villous trophoblasts are crucial for the barrier function of the placenta, this could in part explain placental dysfunction in idiopathic IUGR fetuses.

The simultaneous role of an alveolus as flow mixer and flow feeder for the deposition of inhaled submicron particles.

In an effort to understand the fate of inhaled submicron particles in the small sacs, or alveoli, comprising the gas-exchange region of the lung, we calculated the flow in three-dimensional (3D) rhythmically expanding models of alveolated ducts. Since convection toward the alveolar walls is a precursor to particle deposition, it was the goal of this paper to investigate the streamline maps' dependence upon alveoli location along the acinar tree. On the alveolar midplane, the recirculating flow pattern exhibited closed streamlines with a stagnation saddle point. Off the midplane we found no closed streamlines but nested, funnel-like, spiral, structures (reminiscent of Russian nesting dolls) that were directed towards the expanding walls in inspiration, and away from the contracting walls in expiration. These nested, funnel-like, structures were surrounded by air that flowed into the cavity from the central channel over inspiration and flowed from the cavity to the central channel over expiration. We also found that fluid particle tracks exhibited similar nested funnel-like spiral structures. We conclude that these unique alveolar flow structures may be of importance in enhancing deposition. In addition, due to inertia, the nested, funnel-like, structures change shape and position slightly during a breathing cycle, resulting in flow mixing. Also, each inspiration feeds a fresh supply of particle-laden air from the central channel to the region surrounding the mixing region. Thus, this combination of flow mixer and flow feeder makes each individual alveolus an effective mixing unit, which is likely to play an important role in determining the overall efficiency of convective mixing in the acinus.

Local strain distribution in real three-dimensional alveolar geometries.

Mechanical ventilation is not only a life saving treatment but can also cause negative side effects. One of the main complications is inflammation caused by overstretching of the alveolar tissue. Previously, studies investigated either global strains or looked into which states lead to inflammatory reactions in cell cultures. However, the connection between the global deformation, of a tissue strip or the whole organ, and the strains reaching the single cells lining the alveolar walls is unknown and respective studies are still missing. The main reason for this is most likely the complex, sponge-like alveolar geometry, whose three-dimensional details have been unknown until recently. Utilizing synchrotron-based X-ray tomographic microscopy, we were able to generate real and detailed three-dimensional alveolar geometries on which we have performed finite-element simulations. This allowed us to determine, for the first time, a three-dimensional strain state within the alveolar wall. Briefly, precision-cut lung slices, prepared from isolated rat lungs, were scanned and segmented to provide a three-dimensional geometry. This was then discretized using newly developed tetrahedral elements. The main conclusions of this study are that the local strain in the alveolar wall can reach a multiple of the value of the global strain, for our simulations up to four times as high and that thin structures obviously cause hotspots that are especially at risk of overstretching.

Finite element 3D reconstruction of the pulmonary acinus imaged by synchrotron X-ray tomography.

The alveolated structure of the pulmonary acinus plays a vital role in gas exchange function. Three-dimensional (3D) analysis of the parenchymal region is fundamental to understanding this structure-function relationship, but only a limited number of attempts have been conducted in the past because of technical limitations. In this study, we developed a new image processing methodology based on finite element (FE) analysis for accurate 3D structural reconstruction of the gas exchange regions of the lung. Stereologically well characterized rat lung samples (Pediatr Res 53: 72-80, 2003) were imaged using high-resolution synchrotron radiation-based X-ray tomographic microscopy. A stack of 1,024 images (each slice: 1024 x 1024 pixels) with resolution of 1.4 mum(3) per voxel were generated. For the development of FE algorithm, regions of interest (ROI), containing approximately 7.5 million voxels, were further extracted as a working subunit. 3D FEs were created overlaying the voxel map using a grid-based hexahedral algorithm. A proper threshold value for appropriate segmentation was iteratively determined to match the calculated volume density of tissue to the stereologically determined value (Pediatr Res 53: 72-80, 2003). The resulting 3D FEs are ready to be used for 3D structural analysis as well as for subsequent FE computational analyses like fluid dynamics and skeletonization.

Loss of beta1-integrin-deficient cells during the development of endoderm-derived epithelia.

Beta1-integrins (beta1) represent cell surface receptors which mediate cell-matrix and cell-cell interactions. Fässler and Meyer described chimeric mice containing transgenic cells that express the LacZ gene instead of the beta1 gene. They observed beta1-negative cells in all germ layers at embryonic day E 8.5. Later in development, using a glucose phosphate isomerase assay of homogenized tissue samples, high levels of transgenic cells were found in skeletal muscle and gut, low levels in lung, heart, and kidney and none in the liver and spleen (Fässler and Meyer 1995). In order to study which cell types require beta1 during development of the primitive gut including its derivatives, chimeric fetuses containing 15 to 25% transgenic cells were obtained at days E 14.5 and E 15.5. They were LacZ (beta-galactosidase) stained "en bloc" and cross-sectioned head to tail. In esophagus, trachea, lung, stomach, hindgut, and the future urinary bladder, we observed various mesoderm-derived beta1-negative cells (e.g. fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells) but no beta1-negative epithelial cells. Since the epithelia of lung, esophagus, trachea, stomach, hindgut, and urinary bladder are derived from the endodermal gut tube, we hypothesize that beta1 is essential for the development and/or survival of the epithelia of the fore- and hindgut and its derivatives.

The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions.

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.

Beta1-class integrins regulate the development of laminae and folia in the cerebral and cerebellar cortex.

Mice that lack all beta1-class integrins in neurons and glia die prematurely after birth with severe brain malformations. Cortical hemispheres and cerebellar folia fuse, and cortical laminae are perturbed. These defects result from disorganization of the cortical marginal zone, where beta1-class integrins regulate glial endfeet anchorage, meningeal basement membrane remodeling, and formation of the Cajal-Retzius cell layer. Surprisingly, beta1-class integrins are not essential for neuron-glia interactions and neuronal migration during corticogenesis. The phenotype of the beta1-deficient mice resembles pathological changes observed in human cortical dysplasias, suggesting that defective integrin-mediated signal transduction contributes to the development of some of these diseases.

Use of genetically modified glial cells overexpressing laminin alpha1-chain peptides in neurite outgrowth studies.

1. C6 glioma cells were transfected with two constructs carrying C-terminal laminin alpha1-chain sequences of 117 and 114 bp length, respectively. These sequences are specifically known to code for peptides which have neurite-promoting activity. 2. The stable expression and secretion of the two peptides was detected by Northern and Western blot analysis. 3. Primary neuronal cultures derived from embryonic mouse forebrain were cocultured with these transfected cells and exhibited a substantial increase in neurite outgrowth and in survival time. Conditioned media from the transfected cells generated similar effects. 4. Organotypic cultures from embryonic mouse brain were used as a second system as being closer to the in vivo situation. Again, coculture of brain slices with transfected cells or treatment with laminin peptide-containing media increased neuronal outgrowth.

Spontaneous peristaltic airway contractions propel lung liquid through the bronchial tree of intact and fetal lung explants.

Spontaneous contractions of the fetal airways are a well recognized but poorly characterized phenomenon. In the present study spontaneous narrowing of the airways was analyzed in freshly isolated lungs from early to late gestation in fetal pigs and rabbits and in cultured fetal mouse lungs. Propagating waves of contraction traveling proximal to distal were observed in fresh lungs throughout gestation which displaced the lung liquid along the lumen. In the pseudoglandular and canalicular stages (fetal pigs) the frequency ranged from 2.3 to 3.3 contractions/min with a 39 to 46% maximum reduction of lumen diameter. In the saccular stage (rabbit) the frequency was 10 to 12/min with a narrowing of approximately 30%. In the organ cultures the waves of narrowing started at the trachea in whole lungs, or at the main bronchus in lobes (5.2 +/- 1.5 contractions/min, 22 +/- 8% reduction of lumen diameter), and as they proceeded distally along the epithelial tubes the luminal liquid was shifted toward the terminal tubules, which expanded the endbuds. As the tubules relaxed the flow of liquid was reversed. Thus the behavior of airway smooth muscle in the fetal lung is phasic in type (like gastrointestinal muscle) in contrast to that in postnatal lung, where it is tonic. An intraluminal positive pressure of 2.33 +/- 0.77 cm H(2)O was recorded in rabbit fetal trachea. It is proposed that the active tone of the smooth muscle maintains the positive intraluminal pressure and acts as a stimulus to lung growth via the force exerted across the airway wall and adjacent parenchyma. The expansion of the compliant endbuds by the fluid shifts at the airway tip may promote their growth into the surrounding mesenchyme.

Programmed cell death contributes to postnatal lung development.

The rat lung undergoes the phase of maturation of the alveolar septa and of the parenchymal microvascular network mainly during the third postnatal week. Speculating that programmed cell death may contribute to the thinning of the alveolar septa, we searched for the presence of DNA fragmentation in rat lungs between postnatal days 6 and 36 using the TUNEL procedure. The number of positive nuclei was compared at different days. We observed an 8-fold increase of programmed cell death toward the end of the third week as compared to the days before and after this time point. The precise timing of the appearance of the peak depended on the size of the litter. Double-labeling for DNA fragmentation (TUNEL) and for type I and type II epithelial cells (antibodies E11 and MNF-116), as well as morphologic studies at electron microscopic level, revealed that during the peak of programmed cell death mainly fibroblasts and type II epithelial cells were dying. While both dying cell types were TUNEL-positive, nuclear fragments and apoptotic bodies were exclusively observed in the dying fibroblasts. We conclude that programmed cell death is involved in the structural maturation of the lung by reducing the number of fibroblasts and type II epithelial cells in the third postnatal week. We observed that the dying fibroblasts are cleared by neighboring fibroblasts in a later stage of apoptosis, and we hypothesize that type II epithelial cells are cleared by alveolar macrophages in early stages of the programmed cell death process.

Protein cross-linking mediated by tissue transglutaminase correlates with the maturation of extracellular matrices during lung development.

At birth, the mammalian lung is still immature. The alveoli are not yet formed and the interairspace walls contain two capillary layers which are separated by an interstitial core. After alveolarization (first 2 postnatal weeks in rats) the alveolar septa mature: their capillary layers merge, the amount of connective tissue decreases, and the mature lung parenchyma is formed (second and third week). During the first 3 wk of life the role of tissue transglutaminase (tTG) was studied in rat lung by immunostaining of cryostat and paraffin sections, by Northern and Western blotting, and by a quantitative determination of gamma-glutamyl-epsilon-lysine. While enzyme activity and intracellular tTG were already present before term, the enzyme product (gamma-glutamyl-epsilon-lysine-crosslink) and extracellular tTG appeared between postnatal days 10 and 19 in the lung parenchyma. In large blood vessels and large airways, which mature earlier than the parenchyma, both the enzyme product and extracellular tTG had already appeared at the end of the first postnatal week. We conclude that tTG is expressed and externalized into the extracellular matrix of lung shortly before maturation of an organ area. Because tTG covalently and irreversibly crosslinks extracellular matrix proteins, we hypothesize that it may prevent or delay further remodeling of basement membranes and may stabilize other extracellular components, such as microfibrils.

Immunostaining of a heterodimeric dermatan sulphate proteoglycan is correlated with smooth muscles and some basement membranes.

A heterodimeric 760-kDa dermatan sulphate proteoglycan tentatively named PG-760 was characterized as a product of keratinocytes, endothelial cells, and fibroblasts. The two core proteins of 460 kDa and 300 kDa are linked by disulphide bridges, and both carry one or only very few dermatan sulphate chains. Different antisera against PG-760 were used in the present study to investigate the distribution in selected murine tissues by light and electron microscopy. PG-760 immunostaining was observed in cornea (epithelium including basement membrane, stroma, and Descemet's membrane), skin, mucosa of the small intestine, Engelbreth-Holm-Swarm (EHS)-tumour (matrix and cells), and the smooth muscle layers of uterus, small intestine, and blood vessels. No staining was observed in capillaries, striated muscles, and liver parenchyma including the central vein. The expression of PG-760 in EHS-tumour was also demonstrated after extraction with 4 M guanidine and partial purification by diethylaminoethyl (DEAE)-chromatography. We conclude that this novel proteoglycan exhibits a unique tissue distribution being a constituent of some but not all basement membranes, of some other extracellular matrices, and additionally, of all investigated smooth muscle layers.

Affinity retardation chromatography: characterization of the method and its application. The description of low affinity laminin self-interactions.

Affinity retardation chromatography (ARC), a method for the examination of low-affinity interactions, is mathematically described in order to characterize the method itself and to estimate binding coefficients of self-assembly domains of basement membrane protein laminin. Affinity retardation was determined by comparing the elutions on a "binding" and on a "nonreacting" column. It depends on the binding coefficient, the concentrations of both ligands, and the nonbinding elution position. Half maximal binding of the NH2-terminal domain of laminin B1-short arm to the A- and/or B2-short arms was estimated to occur at 10-17 microM for noncooperative and at < or = 3 microM for cooperative binding. A model of the laminin polymerization, postulating two levels of cooperative binding behavior, is described.

Role of the B1 short arm in laminin self-assembly.

Laminin self-assembles into a basement membrane polymer through specific low-affinity interactions. Recently, it was shown that the terminal short-arm domain (domains VI and V) of the B1 chain (fragment E4) possesses one of the laminin self-interaction sites [Schittny, J.C. & Yurchenco, P.D. (1990) J. Cell Biol. 110, 825-832], but that the binding partner(s) of this domain is unknown. Using affinity retardation chromatography we now investigate the domain(s) fragment E4 binds to. The elution of E4 was clearly retarded on immobilized laminin and fragment E1' (three-chain short-arm complex excluding the distal part of the B1 chain), but not on immobilized E4 in calcium containing buffer and at 37 degrees C. Under the same conditions, E1' strongly interacts with immobilized E4. In addition, E1' is able to non-covalently cross-link soluble E4 to immobilized E4. No further interaction of laminin and E4 with additional fragments (P1', A, B2 and B1 chain short-arm complex without B1-domains VI-IV and without globules; E8, distal long arm and G1-3; E3, long-arm G subdomains 4 and 5) could be demonstrated. These data are interpreted as evidence that (a) the primary laminin-laminin bonds are formed between the short arms of laminin, that (b) the terminal B1 short-arm domain (E4) can interact with the short arm(s) of the A and/or B2 chain(s) (domain E1'), but does not self-interact, and that (c) due to at least three self-binding sites, laminin polymerization behaves co-operatively.

Intussusceptive microvascular growth: a common alternative to capillary sprouting.

Intussusceptive capillary growth represents a new principle for microvascular growth as described in the lungs of growing rats. According to this concept, the capillary network expands by the formation of slender transcapillary tissue pillars, which give rise to new vascular meshes. The process was first observed in Mercox casts of the lung microvasculature, which revealed the existence of multiple tiny holes with diameters around 1.5 microns. Consecutive transmission electron microscopic investigation of serial sections demonstrated that the holes corresponded to slender tissue pillars (Burri and Tarek, 1990). The corrosion cast technique thus appears to be an adequate screening method for intussusceptive growth. In the present investigation, Mercox casts of various vascular systems, namely, those of the eye, submandibular gland, heart, liver, stomach, small and large intestine, trachea, kidney, uterus and ovary were prepared from rats aged between 4 and 9 weeks in order to screen them for the existence of the typical tiny holes representing tissue pillars. In all organs investigated, these structures were observed in various locations to a variable degree. They were mainly encountered within dilated vascular segments or at triple or quadruple branching points of the circulation. Even in capillary networks with a three-dimensional arrangement could these pillars be detected. Intussusception thus appears to be a principle of growth appertaining to many vascular systems.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs.

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Molecular architecture of basement membranes.

Basement membranes are specialized extracellular matrices with support, sieving, and cell regulatory functions. The molecular architectures of these matrices are created through specific binding interactions between unique glycoprotein and proteoglycan protomers. Type IV collagen chains, using NH2-terminal, COOH-terminal, and lateral association, form a covalently stabilized polygonal framework. Laminin, a four-armed glycoprotein, self-assembles through terminal-domain interactions to form a second polymer network, Entactin/nidogen, a dumbbell-shaped sulfated glycoprotein, binds laminin near its center and interacts with type IV collagen, bridging the two. A large heparan sulfate proteoglycan, important for charge-dependent molecular sieving, is firmly anchored in the basement membrane and can bind itself through a core-protein interaction to form dimers and oligomers and bind laminin and type IV collagen through its glycosaminoglycan chains. Heterogeneity of structure and function occur in different tissues, in development, and in response to different physiological needs. The molecular architecture of these matrices may be regulated during or after primary assembly through variations in compositions, isoform substitutions, and the modifying influence of exogenous macromolecules such as heparin and heparan sulfate.

Terminal short arm domains of basement membrane laminin are critical for its self-assembly.

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.

Heparin modulation of laminin polymerization.

Previously, it has been shown that laminin will self-assemble by a two-step calcium-dependent process using end-domain interactions (Yurchenco, P. D., Tsi-library, E. C., Charonis, A. S., and Furthmayr, H. (1985) J. Biol. Chem. 260, 7636-7644). We now find that heparin, at low concentrations, modifies this polymerization by driving the equilibrium further toward aggregation, by producing a denser polymer, and by inducing aggregation in the absence of calcium. This effect on self-assembly is specific in that it is observed with heparin but not with several heparan sulfates or other glycosaminoglycans: it correlates with affinity and depends on the degree of polysaccharide sulfation. Heparin binds to laminin in a calcium-dependent manner with a single class of interaction (KD = 118 +/- 18 nM) and with a binding capacity of one heparin for two laminins. We find the long arm globule (E3) is the only laminin domain which exhibits substantial heparin binding: heparin binds E3 with an affinity (KD = 94 +/- 12 nM) and calcium dependence similar to that for intact laminin. These data strongly suggest that heparin modifies laminin assembly by binding to pairs of long arm globular domains. As a result the polymer may be stabilized at domain E3 and laminin interdomain interactions induced or modified. We further postulate that heparins may act in vivo as specific regulators of the structure and functions of basement membranes by both altering the laminin matrix and by displacing weakly binding heparan sulfates.