Martin Sandig

Connexin 43 mediated gap junctional communication enhances breast tumor cell diapedesis in culture

IntroductionMetastasis involves the emigration of tumor cells through the vascular endothelium, a process also known as diapedesis. The molecular mechanisms regulating tumor cell diapedesis are poorly understood, but may involve heterocellular gap junctional intercellular communication (GJIC) between tumor cells and endothelial cells.MethodTo test this hypothesis we expressed connexin 43 (Cx43) in GJIC-deficient mammary epithelial tumor cells (HBL100) and examined their ability to form gap junctions, establish heterocellular GJIC and migrate through monolayers of human microvascular endothelial cells (HMVEC) grown on matrigel-coated coverslips.ResultsHBL100 cells expressing Cx43 formed functional heterocellular gap junctions with HMVEC monolayers within 30 minutes. In addition, immunocytochemistry revealed Cx43 localized to contact sites between Cx43 expressing tumor cells and endothelial cells. Quantitative analysis of diapedesis revealed a two-fold increase in diapedesis of Cx43 expressing cells compared to empty vector control cells. The expression of a functionally inactive Cx43 chimeric protein in HBL100 cells failed to increase migration efficiency, suggesting that the observed up-regulation of diapedesis in Cx43 expressing cells required heterocellular GJIC. This finding is further supported by the observation that blocking homocellular and heterocellular GJIC with carbenoxolone in co-cultures also reduced diapedesis of Cx43 expressing HBL100 tumor cells.ConclusionCollectively, our results suggest that heterocellular GJIC between breast tumor cells and endothelial cells may be an important regulatory step during metastasis.

Differential regulation of transendothelial migration of THP-1 cells by ICAM-1/LFA-1 and VCAM-1/VLA-4.

The adhesion molecules intercellular adhesion molecule 1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1) expressed in atherogenic lesions are thought to regulate monocyte diapedesis. To better understand their specific roles we used function‐blocking antibodies and examined in a culture model the morphology, motility, and diapedesis of THP‐1 cells interacting with human coronary artery endothelial cells. The number of motile THP‐1 cells was reduced only when VCAM‐1 or both ICAM‐1 and VCAM‐1 were blocked. Blockade of ICAM‐1 and VCAM‐1, either separately or together, reduced to the same degree the distance that THP‐1 cells traveled. Diapedesis was reduced only during the simultaneous blockade of both adhesion molecules. Blockade of either ICAM‐1 or VCAM‐1 inhibited pseudopodia formation, but ICAM‐1 blockade induced the formation of filopodia. We suggest that the interactions of endothelial ICAM‐1 and VCAM‐1 with their ligands differentially regulate distinct steps of diapedesis by modulating the ratio of active and inactive forms of small GTPases such as Rho, Rac, and Cdc42.

Neutrophils Induce Sequential Focal Changes in Endothelial Adherens Junction Components: Role of Elastase

Objective: In vitro studies have indicated that polymorphonuclear leukocytes (PMNs) traverse endothelial cell monolayers via the paracellular pathway (i.e., through endothelial cell–cell junctions. Herein, we assessed whether the adherens junctions (AJs) are disrupted during PMN transendothelial cell migration.

The p110δ Isoform of PI3K Differentially Regulates β1 and β2 Integrin-Mediated Monocyte Adhesion and Spreading and Modulates Diapedesis

Objective: Leukocyte diapedesis is misregulated in inflammatory disease and depends on the binding of monocytic LFA‐1 and VLA‐4 to endothelial ICAM‐1 and VCAM‐1, respectively. The authors hypothesized that these different molecular interactions elicit specific signaling cascades within monocytes regulating specific steps in adhesion, motility, and diapedesis.

Characterization of cell elasticity correlated with cell morphology by atomic force microscope

Biomechanical properties of cells have been identified as an important factor in a broad range of biological processes. Based on measurements of mechanical properties by atomic force microscopy (AFM) particularly cell elasticity has been linked with human diseases, such as cancer. AFM has been widely used as a nanomechanical tool to probe the elasticity of living cells, however, standard methods for characterizing cell elasticity are still lacking. The local elasticity of a cell is conventionally used to represent the mechanical property of the cell. However, since cells have highly heterogeneous regions, elasticity mapping over the entire cell, rather than at a few points of measurement, is required. Using human aortic endothelial cells (HAECs) as a model, we have developed in this study a new method to evaluate cell elasticity more quantitatively. Based on the height information of the cell, a new characterization method was proposed to evaluate the elasticity of a cell. Using this method, elasticities of cells on different substrates were compared. Results showed that the elasticity of HAECs on softer substrate also has higher value compared to those on harder substrate given a certain height where the statistical distribution analysis confirmed that higher actin filaments density was located. Thus, the elasticity of small portions of a cell could not represent the entire cell property and may lead to invalid characterization. In order to gain a more comprehensive and detailed understanding of biomechanical properties for future clinical use, elasticity and cell morphology should therefore be correlated with discussion.

Interactions of coronary artery smooth muscle cells with 3D porous polyurethane scaffolds.

One strategy in vascular tissue engineering is the design of hybrid vascular substitutes where vascular cells infiltrate biostable porous scaffolds that provides favorable environment for guided cell repopulation and acts as a mechanically supporting layer after the tissue regeneration process. The aim of the present work was to study the interaction of human coronary artery smooth muscle cells (HCASMC) with 3D porous polyurethane scaffolds. We therefore fabricated porous and highly interconnected 3D polyurethane scaffolds that can promote HCASMC attachment, proliferation, and migration. SEM and microCT studies of the fabricated scaffolds showed that the current scaffolds had highly open and interconnected pore structures, with an average porosity of 84%. HCASMC interaction on polyurethane films revealed that cells adhere and express specific marker proteins (vinculin and h-caldesmon). This expression was further enhanced by coating the polyurethane with Matrigel. On uncoated 3D scaffolds, dense spherical aggregates of cells were often encountered with little adhesion of individual cells alongside the struts of the scaffold, independent of the porogens used. In contrast, when cultured on Matrigel-coated scaffolds, cell numbers quickly increased after 14 days and spread along the entire scaffold. At the upper scaffold surface, elongated cells were seen adhering to one another and also to the scaffold surface. These cells were elongated, aligned in parallel and contained abundant F-actin bundles suggesting a differentiated contractile phenotype. Deep into the scaffold, cells were encountered that formed actin-rich lamellipodial extensions spreading along the strut and lacked stress fibers, suggesting active cell migration along the substrate.

Interleukin-1β Reduces Transcellular Monocyte Diapedesis and Compromises Endothelial Adherens Junction Integrity

Objective: Diapedesis occurs through endothelial cell–cell junctions (paracellular) or through individual endothelial cells without disrupting junctions (transcellular). While in vitro studies have provided considerable insight into mechanisms controlling paracellular diapedesis, little is known about what regulates transcellular diapedesis. The authors investigated whether transcellular diapedesis is susceptible to IL‐1β exposure of the endothelium.

Transendothelial Migration of Monocytes in Rat Aorta: Distribution of F-actin, α-Catenin, LFA-1, and PECAM-1

To determine changes in the distribution of cell adhesion molecules during diapedesis of monocytes in situ, we labeled aortic whole mounts from hypercholesterolemic rats with Texas red-phalloidin and antibodies to LFA-1, PECAM-1, or α-catenin, and analyzed them by laser scanning confocal microscopy. Monocytes transmigrated through circular openings (transmigration passages) formed by pseudopodia that penetrated between adjacent en-dothelial cells. Transmigrating monocytes remained spherical above the endothelium, while spreading beneath it. The transmigration passage was lined by F-actin and partially by α-catenin, suggesting cadherin-mediated heterotypic interactions. LFA-1 was present in clusters at the monocyte cell surface throughout diapedesis, but was concentrated at the margin of the transmigration passage. PECAM-1 was enriched in the endothelial contact regions where the monocytes transmigrated. PECAM-1 was barely detectable in monocytes before and after diapedesis, but appeared during diapedesis ...

The role of endothelial cell-bound Jagged1 in Notch3-induced human coronary artery smooth muscle cell differentiation

Phenotype regulation of vascular smooth muscle cells (VSMC) is an important requirement in both tissue engineering and balloon angioplasty strategies. In recent years, it has become increasingly evident that the Notch signalling pathway plays a critical role in regulating vascular morphogenesis during development and the transcription of differentiated VSMC and its maturation. In arteries, Notch3 is the predominant receptor on VSMC and, signalling is initiated upon binding to its ligand, Jagged1. However, little is known on how ligand presenting strategies affect Notch signalling and subsequently upregulation of smooth muscle cell differentiation. In this study, using human coronary artery smooth muscle cells (HCASMC) and human coronary artery endothelial cells (HCAEC), we show several lines of evidence that direct heterocellular cell-cell contact is necessary for VSMC differentiation via Notch3 signalling. First, neither the addition of soluble Jagged1 nor Jagged1 immobilized to protein G beads induced HCASMC differentiation in culture. Second, despite the upregulation of Notch3 expression, EC-conditioned medium failed to induce HCASMC differentiation. However, when HCASMC and HCAEC were co-cultured either on opposite sides of porous membrane or when these cells were co-cultured directly, both Notch3 and VSMC differentiation marker proteins were upregulated. These upregulations were abrogated by Jagged1-specific siRNA. This study provides the first direct evidence that contact of HCASMC and HCAEC is required for regulating smooth muscle cell differentiation. These findings may have clinical importance and therapeutic potential for modulating vascular SMC phenotype during various cardiovascular disease states and in tissue engineering.

The development of a virtual 3D model of the renal corpuscle from serial histological sections for E-learning environments.

Histology is a core subject in the anatomical sciences where learners are challenged to interpret two‐dimensional (2D) information (gained from histological sections) to extrapolate and understand the three‐dimensional (3D) morphology of cells, tissues, and organs. In gross anatomical education 3D models and learning tools have been associated with improved learning outcomes, but similar tools have not been created for histology education to visualize complex cellular structure–function relationships. This study outlines steps in creating a virtual 3D model of the renal corpuscle from serial, semi‐thin, histological sections obtained from epoxy resin‐embedded kidney tissue. The virtual renal corpuscle model was generated by digital segmentation to identify: Bowmans capsule, nuclei of epithelial cells in the parietal capsule, afferent arteriole, efferent arteriole, proximal convoluted tubule, distal convoluted tubule, glomerular capillaries, podocyte nuclei, nuclei of extraglomerular mesangial cells, nuclei of epithelial cells of the macula densa in the distal convoluted tubule. In addition to the imported images of the original sections the software generates, and allows for visualization of, images of virtual sections generated in any desired orientation, thus serving as a “virtual microtome”. These sections can be viewed separately or with the 3D model in transparency. This approach allows for the development of interactive e‐learning tools designed to enhance histology education of microscopic structures with complex cellular interrelationships. Future studies will focus on testing the efficacy of interactive virtual 3D models for histology education. Anat Sci Educ 8: 574–583.