Intercellular Boundaries Research Articles

Strength stability at high temperatures for additively manufactured alumina forming austenitic alloy

Several fast-spectrum nuclear reactors designed to generate high power (∼450 MWe) rely on forced convection of media such as supercritical CO2, sodium, or liquid lead to cool the nuclear core, operating at temperatures up to 600 °C. Cost-effective, high-strength Fe-based alumina forming austenitic (AFA) alloys are a promising candidate for the fabrication of critical nuclear components. This study investigated laser powder bed fusion (LPBF) processing of an AFA alloy composition optimized for improved creep resistance. Electron microscopy revealed an elongated grain structure along the build direction with a fine sub-grain cellular structure decorated with (Cr,Fe,Nb)23C6 carbide precipitates at the intercellular boundaries. At temperatures of 20–900 °C, the LPBF alloy's superior tensile properties compared to its arc-melted counterpart and other advanced steels (e.g., SS316) were attributed to the distribution of nano-sized carbide precipitates, whereas the high ductility was attributed to the LPBF alloy's elongated grain structure.

Topological balance of cell distributions in plane monolayers

Most of normal proliferative epithelia of plants and metazoans are topologically invariant and characterized by similar cell distributions according to the number of cell neighbors (DCNs). Here we study peculiarities of these distributions and explain why the DCN obtained from the location of intercellular boundaries and that based on the Voronoi tessellation with nodes located on cell nuclei may differ from each other. As we demonstrate, special microdomains where four or more intercellular boundaries converge are topologically charged. Using this fact, we deduce a new equation describing the topological balance of the DCNs. The developed theory is applied for a series of microphotographs of non-tumoral epithelial cells of the human cervix (HCerEpiC) to improve the image processing near the edges of microphotographs and reveal the topological invariance of the examined monolayers. Special contact microdomains may be present in epithelia of various natures, however, considering the well-known vertex model of epithelium, we show that such contacts are absent in the usual solid-like state of the model and appear only in the liquid-like cancer state. Also, we discuss a possible biological role of special contacts in context of proliferative epithelium dynamics and tissue morphogenesis.

Adenylate cyclase toxin of Bordetella parapertussis disrupts the epithelial barrier granting the bacterial access to the intracellular space of epithelial cells.

B. parapertussis is one of the etiological agents of whooping cough. Once inhaled, the bacteria bind to the respiratory epithelium and start the infection. Little is known about this first step of host colonization and the role of the human airway epithelial barrier on B. parapertussis infection. We here investigated the outcome of the interaction of B. parapertussis with a polarized monolayer of respiratory epithelial cells. Our results show that B. parapertussis preferentially attaches to the intercellular boundaries, and causes the disruption of the tight junction integrity through the action of adenylate cyclase toxin (CyaA). We further found evidence indicating that this disruption enables the bacterial access to components of the basolateral membrane of epithelial cells to which B. parapertussis efficiently attaches and gains access to the intracellular location, where it can survive and eventually spread back into the extracellular environment. Altogether, these results suggest that the adenylate cyclase toxin enables B. parapertussis to overcome the epithelial barrier and eventually establish a niche of persistence within the respiratory epithelial cells.

Assessment of the effectiveness of restrictive epidemic control measures using original models of cellular automaton

Background. The ongoing COVID-19 pandemic, the human casualties caused by it, and the possibility of new epidemical threats make the search for effective countermeasures actual. One of the most effective tools, as the experience of the COVID-19 pandemic has shown, is restrictive measures of various types, which are especially significant with medical countermeasures being unavailable or insufficient. At the same time, the topic of restrictive measures and their mathematical modeling, especially given its importance, is not sufficiently disclosed in the scientific literature.The aim. To determine the possibility of assessing the effectiveness of restrictive epidemic control measures using original models of cellular automaton with intercellular boundaries.Methods. To determine the impact of restrictive measures on the dynamics of the daily increase in infected people, an original cellular automaton with intercellular boundaries was developed, which makes it possible to simulate epidemic control measures of varying stringency. In the simulations carried out using the Monte Carlo method with subsequent statistical processing, we studied the impact of restrictive measures of varying stringency on the number of infected people, the duration of the epidemic, and the quality of forecasting. The final series of experiments simulated the spread of the COVID-19 virus in Germany in the first half of 2020.The results show that even a simple cellular automaton model with boundaries successfully describes the course of the epidemic and allows us to assess the effectiveness of restrictive measures. The dependence of the daily increase in infected people on the stringency of measures is presented; it is shown what characteristics of the population can influence this dependence. It was found that the measures of medium stringency (40–50 % according to the Stringency Index) have the least predictable effect; they can cause both rapid localization of the focus and the spread of the epidemic to a large part of the population. Weak and strong measures give a more predictable effect.Conclusion. Cellular automaton models with intercellular boundaries have great potential for modeling the impact of restrictive measures on the course of an epidemic, making it possible to predict the dynamics of infected people based on the population data and the restrictive measures being introduced.

The Microstructural Evolution of CM247LC Manufactured Through Laser Powder Bed Fusion

Numerous challenges persist with the additive manufacturing of high γ′ containing Ni-based superalloys such as CM247LC. Currently, significant cracking occurs during deposition of CM247LC components using laser powder bed fusion and during post-processing. Whilst post-deposition procedures seek to eliminate or minimise cracks, current procedures do not produce a microstructure suitable for service. This study systematically investigates the microstructural evolution of CM247LC manufactured using laser powder bed fusion following multiple post processing treatments. Phase and textural changes after each processing step were consistent with previous studies, although an additional Hf-rich and Cr-depleted segregation zone was identified along intercellular boundaries in the as-deposited condition, believed to be associated with the cracking propensity. Compositional modification of CM247LC including removal of Hf, reduction of C and addition of Nb eliminated the segregation zone but these changes were associated with an increased susceptibility to solidification and liquation cracking.

Effects of handedness ratio and handedness distribution on the mechanical properties of Bi-chiral mechanical metamaterials

The elastic mechanical properties of bi-chiral mechanical metamaterials are explored via systematic finite element simulations. Designs of bi-chiral mechanical metamaterials with both periodic and complete random handedness distributions for various handedness ratio and domain size and length aspect ratios were investigated. It is found that in general, because of the interactions at the ‘twinned’ intercellular boundaries between neighboring domains, the handedness ratio, handedness distribution, domain size and length aspect ratio significantly influence the overall mechanical properties of bi-chiral designs. Specifically, when handedness ratio increases and domain size decreases, the effective stiffness and the auxetic effects increases, while the effective shear modulus is much less sensitive to either the handedness ratio or the domain size. The chirality-induced normal shear coupling effect is solely determined by the handedness ratio. An empirical mechanical model was proposed to quantify the influences of ‘twinned’ intercellular boundaries on the effective stiffness and effective Poisson’s ratio of bi-chiral designs. In addition, both upper and lower limits of the effective stiffness and the effective Poisson’s ratio of bi-chiral designs are identified.

High strength aluminum-cerium alloy processed by laser powder bed fusion

Readily available high strength Al-alloys for laser power bed fusion (LPBF) typically require post-processing such as mechanical working or heat treatment to evolve strengthening phases. Through the calculation of phase diagrams (CALPHAD) and a method based on Scheil solidification, the Al-8 wt%Ce-10 wt%Mg alloy was evaluated for LPBF to be microstructurally favorable to yield components with full volumetric density, without the need for post-processing. Through an exhaustive LPBF parametric study, documentation of defects was performed for the use of a wide range of laser power and scan speed combinations, i.e., varying the energy density, followed by microstructural characterization with X-ray diffraction, optical and electron microscopy, and mechanical testing with both Vickers hardness and quasi-static uniaxial tension. Overall, defects were observed with use of high and low laser powers and scan speeds, but a processing window for LPBF was observed at both high and low laser powers, where > 99% volumetric density was achieved. Besides defects, decreasing the scan speed, i.e., increasing the energy density, resulted in decreases in the concentration of Mg, down to a maximum of 4.5 wt% due to vaporization. Moreover, a decrease in Mg led to a lower Vickers hardness, attributed to a decrease in the solid solutioning of Mg in the α-Al matrix. Samples selected for tensile mechanical testing were produced with a LPBF laser power of 200 W traversing at 800 mm/s, which yielded a density > 99% and Mg concentration of approximately 7 wt%. A yield strength and ultimate tensile strength of 377 and 468 MPa, respectively, was found for those produced with the power of 200 W. However, the ductility of the alloy was less than 2% elongation at failure. A fine (< 1 µm) sub-grain cellular solidification structure was found distributed throughout the α-Al matrix, with ribbons of the eutectic Al11Ce3 intermetallic distributed along the intercellular boundaries. The high strengths achieved in this alloy were attributed to contribution from Orowan dislocation looping of the Al11Ce3 phase, the high degree of solid solutioning of Mg in the α-Al matrix, and cellular boundary strengthening due to impeding dislocation movement.

Folliculin haploinsufficiency causes cellular dysfunction of pleural mesothelial cells

Birt–Hogg–Dubé syndrome (BHDS), an autosomal dominant inheritance disease caused by folliculin (FLCN) mutations, is associated with lung cysts and spontaneous pneumothorax. The possibility of FLCN haploinsufficiency in pleural mesothelial cells (PMCs) contributing to development of pneumothorax has not yet been clarified. Electron microscopy revealed exposed intercellular boundaries between PMCs on visceral pleura and decreased electron density around the adherens junctions in BHDS. To characterize cellular function of PMCs in BHDS patients (BHDS-PMCs), during surgery for pneumothorax, we established the flow cytometry-based methods of isolating high-purity PMCs from pleural lavage fluid. BHDS-PMCs showed impaired cell attachment and a significant decrease in proliferation and migration, but a significant increase in apoptosis compared with PMCs from primary spontaneous pneumothorax (PSP) patients (PSP-PMCs). Microarray analysis using isolated PMCs revealed a significant alteration in the expression of genes belonging to Gene Ontology terms “cell–cell adhesion junction” and “cell adhesion molecule binding”. Gene set enrichment analysis demonstrated that CDH1, encoding E-cadherin, was identified in the down-regulated leading edge of a plot in BHDS-PMCs. AMPK and LKB1 activation were significantly impaired in BHDS-PMCs compared with PSP-PMCs. Our findings indicate that FLCN haploinsufficiency may affect the E-cadherin-LKB1-AMPK axis and lead to abnormal cellular function in BHDS-PMCs.

Composition-dependent solidification cracking of aluminum-silicon alloys during laser powder bed fusion

Consistent manufacturing of volumetrically dense engineering components, free of solidification cracks by laser powder bed fusion (LPBF), has been demonstrated for Al-Si alloys such as AlSi10Mg and Al12Si. The success in LPBF of these alloys is attributed to the near eutectic composition with a small freezing range. To illuminate this observation, cracking susceptibility was examined from Scheil-Gulliver solidification modeling by calculating the hot cracking susceptibility, |dT/dfS1/2|. To validate the findings from hot cracking susceptibility calculations, six binary Al-Si alloys, whose compositions were strategically chosen at hypo-, near-, and hyper-eutectic compositions, were gas atomized into alloy powders, and processed by LPBF. Only Al-Si alloys with 1.0 and 2.0 wt.% Si were found to exhibit cracking, which was predicted by relatively large magnitudes of |dT/dfS1/2|. Either as particles or with a eutectic structure, Si segregation at the intercellular boundaries was observed to define the sub-grain cellular structure. For selected compositions, measurement of the cellular structure allowed for estimation of the cooling rate to be 106 to 107 K•s−1. Excluding the alloys with solidification cracking, an increase in tensile strength and the corresponding decrease in ductility were observed with an increase in Si concentration, which were attributed to the formation of a cellular structure and the amount of Al-Si eutectic found at the intercellular boundaries.

Resolving tractions across cell‐cell adhesion reveals the role of intercellular shear in plithotaxis

BackgroundEndothelial cells form a selectively permeable layer by attaching to their neighbors through various adhesion molecules. Some of these molecules are interlinked through cytoskeletal filaments and exposed to cytoskeletal forces. Importantly, cytoskeletal forces are balanced continuously at the cell‐cell and cell‐substrate adhesion maintaining monolayer homeostasis and preventing cellular motion. Using Monolayer Stress Microscopy, we have previously quantified cytoskeletal forces across cellular monolayers. We identified that locations with orientation‐dependent (or anisotropic) tension exhibited motion along the orientation of highest tension (or equivalently, the orientation of minimal shear forces). This phenomenon, known as plithotaxis, would occur if the intercellular junctions are unable to support shear forces. Here we examine the validity of this structural basis for plithotaxis in cultured cell monolayers.MethodsWe seeded clusters of pulmonary endothelial cells on a collagen‐coated hydrogel and quantified the cell‐substrate forces by analyzing the motion of beads embedded within the gel. Using these forces, we analyzed mechanical equilibrium of the cell monolayer and recovered a two‐dimensional monolayer stress tensor (or cytoskeletal forces). By segmenting phase contrast image of the cell monolayer, we detected the location of the intercellular boundaries. The monolayer stress tensor at the intercellular boundaries was then resolved into Fn and Ft. Using these forces, we then computed a two‐dimensional moment tensor for each cell. This moment tensor is then used in assessing a novel definition of plithotaxis, not as a property of discrete points but instead, as a property of each cell within the monolayer.ResultsFn and Ft exhibited unique characteristics for endothelial cells from distinct pulmonary vascular segments, including arteries (PAEC), microvessels (PMVEC) and veins (PVEC). The highest contrast was seen between PAECs and PMVECs. PMVECs exhibited the fewest intercellular segments with negative Fn, Ft was largest and most comparable in magnitude to Fn, and the cells appeared to move along the principal axis of the moment tensor, indicating that intercellular shear forces are not negligible and plithotaxis can be defined at the lengthscale of a cell. Moreover, plithotaxis was more pronounced for the cells that had higher median Ft/Fn, indicating that intercellular shear force and plithotaxis are in fact positively correlated.ConclusionPlithotaxis arise not from inability but instead from the strong ability of intercellular junctions to support shear forces. Our work opens new research avenues for systematic examination of the relationship between structure and physical function of the intercellular junctions.Support or Funding InformationAbraham Mitchell Cancer Research Fund, Research and Scholarly Development Grant, P01 HL66299 (NIH/NHLBI), F32 HL144040‐01 (NHLBI), University of South Alabama Honors College Scholarship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FEATURES OF MORPHOFUNCTIONAL REMODELLING OF RAT HEPATOCYTES AND THEIR QUANTITATIVE CHARACTERISTICS AGAINST THE GROUND OF CHRONIC ACETYLSALICYLIC ACID POISONING

According to the results of the study, it has been established that chronic acetylsalicylic acid poisoning results in severe liver disorders presented by both venous and portal blood overloaded with a reflex throughput decrease of the arterial part of the bloodstream. Prolonged venous stasis and reflex vasoconstriction of arteries lead to ischemia of the liver tissue with the development and progression of dystrophic changes in hepatocytes. With an increase in the duration of observation, there is an expansion of cell zones with signs of granular dystrophy, which pass into fields with dystrophy of hepatic cells, damage of intercellular boundaries, homogenization of cytoplasm and degradation of nuclei of hepatocytes. It was manifested quantitatively by an increase in the cross-section of hepatocytes and their nuclei with an appropriate dynamics of nuclear-cytoplasmic relations.

The Influence of Milling and Spark Plasma Sintering on the Microstructure and Properties of the Al7075 Alloy.

The compact samples of an Al7075 alloy were prepared by a combination of gas atomization, high energy milling, and spark plasma sintering. The predominantly cellular morphology observed in gas atomized powder particles was completely changed by mechanical milling. The continuous-like intermetallic phases present along intercellular boundaries were destroyed; nevertheless, a small amount of Mg(Zn,Cu,Al)2 phase was observed also in the milled powder. Milling resulted in a severe plastic deformation of the material and led to a reduction of grain size from several µm into the nanocrystalline region. The combination of these microstructural characteristics resulted in abnormally high microhardness values exceeding 300 HV. Consolidation through spark plasma sintering (SPS) resulted in bulk samples with negligible porosity. The heat exposition during SPS led to precipitation of intermetallic phases from the non-equilibrium microstructure of both gas atomized and milled powders. SPS of the milled powder resulted in a recrystallization of the severely deformed structure. An ultra-fine grained structure (grain size close to 500 nm) with grains divided primarily by high-angle boundaries was formed. A simultaneous release of stored deformation energy and an increase in the grain size caused a drop of microhardness to values close to 150 HV. This value was retained even after annealing at 425 °C.

Surface modification of Al–Si alloy by excimer laser pulse processing

The laser irradiation on Al-Si alloy sample is carried out by excimer laser in ambient conditions for 30 or 45 pulses. Microstructural investigation of laser treated sample is done by OM, SEM and TEM and the surface hardness is evaluated by Vickers micro indentation. Laser treated, samples suggested the dissolution of coarse primary Si and β-AlFeSi particle in α-Al matrix. The SEM/EDS study shows the enhancement of retained Si in α-Al matrix. The interface analysis of laser treated sample suggested the effected modified depth is ∼6 μm. TEM investigation shows the formation of nanocrystalline Si in size ∼2–15 nm. The cellular structures of size range ∼30–50 nm are observed after 45 pulses. The α-Al cells and Si precipitates sizes were considerably refined at higher number of pulses. The fine Si precipitates are found to be dispersed in the intercellular boundaries. An improvement in surface hardness from ∼1.6 to 1.8 is observed 30 and 45 pulse treatment, respectively. The mechanism involves for improvement in surface properties are non-equilibrium solidification, metastable phase formation and microstructural refinement.

The mechanical properties of plant cell walls soft material at the subcellular scale: the implications of water and of the intercellular boundaries

Subcellular mechanical characterization of the cell wall can provide important insights into the cell wall’s functional organization, especially if the characterization is not confounded by extracellular factors and intercellular boundaries. However, due to the technical challenges associated with the microscale mechanical characterization of soft biological materials, subcellular investigations of the plant cell wall under tensile loading have yet to be properly performed. This study reports the mechanical characterization of primary onion epidermal cell wall profiles using a novel cryosection-based sample preparation method and a microelectromechanical system-based tensile testing protocol. At the subcellular scale, the cell wall showed biphasic behavior similar to tissue samples. However, instead of a transition zone between the linear elastic or viscoelastic and linear plastic zones, the subcellular-scale samples showed a plateau-like trend with a sharp drop in the modulus value. The critical ranges of stress (20–40 MPa) and strain (5–12 %) of the plateau zone were identified. A strain energy of 1.3 MJ m−3 was calculated at the midpoint of the critical stress–strain range; this value was in accordance with the previously estimated hydrogen bond energy of the cell wall. Subcellular-scale samples showed very large lateral/axial deformations (0.8 ± 0.13) at fracturing. In addition, investigating the cell wall’s mechanical properties at three different water states showed that water is critical for the flow-like behavior of cell wall matrix polymers. These results at subcellular scale provide new insights into biological materials that possess a structural hierarchy at different length scales; which cannot be obtained from tissue-scale experiments.

ADAM12-directed ectodomain shedding of E-cadherin potentiates trophoblast fusion.

Trophoblasts, placental cells of epithelial lineage, undergo extensive differentiation to form the cellular components of the placenta. Trophoblast progenitor cell differentiation into the multinucleated syncytiotrophoblast is a key developmental process required for placental function, where defects in syncytiotrophoblast formation and turnover associate with placental pathologies and link to poor pregnancy outcomes. The cellular and molecular processes governing syncytiotrophoblast formation are poorly understood, but require the activation of pathways that direct cell fusion. The protease, A Disintegrin and Metalloproteinase 12 (ADAM12), controls cell fusion in myoblasts and is highly expressed in the placenta localizing to multiple trophoblast populations. However, the importance of ADAM12 in regulating trophoblast fusion is unknown. Here, we describe a function for ADAM12 in regulating trophoblast fusion. Using two distinct trophoblast models of cell fusion, we show that ADAM12 is dynamically upregulated and is under the transcriptional control of protein kinase A. siRNA-directed loss of ADAM12 impedes spontaneous fusion of primary cytotrophoblasts, whereas overexpression of the secreted variant, ADAM12S, potentiates cell fusion in the Bewo trophoblast cell line. Mechanistically, both ectopic and endogenous levels of ADAM12 were shown to control trophoblast fusion through E-cadherin ectodomain shedding and remodeling of intercellular boundaries. This study describes a novel role for ADAM12 in placental development, specifically highlighting its importance in controlling the differentiation of villous cytotrophoblasts into multinucleated cellular structures. Moreover, this work identifies E-cadherin as a novel ADAM12 substrate, and highlights the significance that cell adhesion molecule ectodomain shedding has in normal development.

Cell Detection From Redundant Candidate Regions Under Nonoverlapping Constraints

Cell detection in microscopy images is essential for automated cell behavior analysis including cell shape analysis and cell tracking. Robust cell detection in high-density and low-contrast images is still challenging since cells often touch and partially overlap, forming a cell cluster with blurry intercellular boundaries. In such cases, current methods tend to detect multiple cells as a cluster. If the control parameters are adjusted to separate the touching cells, other problems often occur: a single cell may be segmented into several regions, and cells in low-intensity regions may not be detected. To solve these problems, we first detect redundant candidate regions, which include many false positives but in turn very few false negatives, by allowing candidate regions to overlap with each other. Next, the score for how likely the candidate region contains the main part of a single cell is computed for each cell candidate using supervised learning. Then we select an optimal set of cell regions from the redundant regions under nonoverlapping constraints, where each selected region looks like a single cell and the selected regions do not overlap. We formulate this problem of optimal region selection as a binary linear programming problem under nonoverlapping constraints. We demonstrated the effectiveness of our method for several types of cells in microscopy images. Our method performed better than five representative methods, achieving an F-measure of over 0.9 for all data sets. Experimental application of the proposed method to 3-D images demonstrated that also works well for 3-D cell detection.

Morphological alterations in the synganglion and integument of Rhipicephalus sanguineus ticks exposed to aqueous extracts of neem leaves (Azadirachta indica A. JUSS).

Currently, the necessity of controlling infestation by ticks, especially by Rhipicephalus sanguineus, has led researchers and public health managers around the world to search for new and more efficient control methods. This way, we can highlight neem (Azadirachta indica A. Juss) leaf, bark, and seed extracts, which have been very effective on tick control, and moreover causing less damage to the environment and to the host. This study showed the potential of neem as a control method for R. sanguineus through morphological and morphometric evaluation of the integument and synganglion of females, in semiengorged stage. To attain this, routine techniques of optical microscopy, scanning electron microscopy and morphometry of the cuticle and subcuticle of the integument were applied. Expressive morphological alterations were observed in both organs, presenting a dose-dependent effect. Integument epithelial cells and nerve cells of the synganglion showed signs of cell vacuolation, dilated intercellular boundaries, and cellular disorganization, alterations not previously reported in studies with neem. In addition, variations in subcuticle thickness were also observed. In general, the effects of neem are multiple, and affect the morphology and physiology of target animals in various ways. The results presented in this work are the first evidence of its effects in the coating and nervous system of ticks, thus allowing an indication of neem aqueous extracts as a potential control method of the brown dog tick and opening new perspectives on acaricide use.

Investigating the machinability of austempered ductile irons with dual matrix structures

Abstract In this study, the machinability of austempered ductile iron with dual phase structure was investigated. Specimens were intercritically austenitized (partially austenitized) in the two phase region (α + γ) at various temperatures (810, 820 and 830 °C) for 90 min, then quenched in a salt bath held at austempering temperatures of 315 °C or 375 °C for 120 min and then air cooled to room temperature to obtain various ausferrite volume fractions and morphologies. Machinability tests were carried out at various cutting speeds (150, 180 and 210 m min−1), at a constant feed rate (0.30 mm rev−1) and depth of cut at 2 mm after austempering heat treatment. The experimental results showed that austempered specimens from the (α + γ) temperature range exhibited better machinability than as-cast samples. On the other hand, the cutting force was decreased with increasing proeutectoid ferrite fractions. The results also showed that continuity of ausferritic structure along intercellular boundaries plays an important role in determining machinability of austempered ductile iron with different ausferrite volume fractions. Another important result is that higher proeutectoid volume fraction has negative effects on surface roughness.

Observation of columnar microstructure in lattice-matched InAlN/GaN grown by plasma assisted molecular beam epitaxy

Nominally lattice matched InAlN/GaN was grown by plasma-assisted molecular beam epitaxy, and the intrinsic microstructure was investigated via x-ray diffraction, transmission electron microscopy, and atom probe tomography. The InAlN showed a cellular structure, which was comprised of ∼10 nm wide Al-rich cores and ∼1 nm In-rich InAlN intercellular boundaries. Despite the strong laterally non-uniform In distribution, both vertical and lateral lattices are unperturbed by the cellular structure, as evidenced by strong thickness fringes in on-axis ω−2θ high resolution x-ray diffraction scans, coherence lengths derived from on-axis (0002) and off-axis (101¯2) ω−2θ high resolution x-ray diffraction scans, and a modified Williamson-Hall analysis for on-axis reflections.

Thrombospondin-1 opens the paracellular pathway in pulmonary microvascular endothelia through EGFR/ErbB2 activation.

Thrombospondin-1 (TSP1) is a multidomain protein that contains epidermal growth factor (EGF)-like repeats that indirectly activate the EGF receptor (EGFR) and selected downstream signaling pathways. In these studies, we show that TSP1 opens the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls) in a dose-, time-, and protein tyrosine kinase (PTK)-dependent manner. TSP1 increased tyrosine phosphorylation of proteins enriched to intercellular boundaries including the zonula adherens (ZA) proteins, vascular endothelial-cadherin, γ-catenin, and p120 catenin. In HMVEC-Ls, EGFR and ErbB2 are expressed at low levels, and both heterodimerize and tyrosine autophosphorylate in response to TSP1. Prior EGFR-selective PTK inhibition with AG1478 or ErbB2-selective PTK inhibition with AG825 protected against TSP1-induced tyrosine phosphorylation of ZA proteins and barrier disruption. Preincubation of HMVEC-Ls with an EGFR ectodomain-blocking antibody also prevented TSP1-induced opening of the paracellular pathway. Therefore, in HMVEC-Ls, TSP1 increases tyrosine phosphorylation of ZA proteins and opens the paracellular pathway, in part, through EGFR/ErbB2 activation. Surprisingly, recombinant TSP1 EGF-like repeats 1-3 and the high-affinity EGFR ligands, EGF, TGF-α, and amphiregulin, each failed to increase paracellular permeability. However, HMVEC-Ls in which EGFR was overexpressed became responsive to the EGF-like repeats of TSP1 as well as to EGF. These studies indicate that TSP1 disrupts the endothelial barrier through EGFR/ErbB2 activation although additional signals are necessary in cells with low receptor expression.