Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle.

Efforts to promote sprouting angiogenesis in skeletal muscles of individuals with peripheral artery disease have not been clinically successful. We discovered that, contrary to the prevailing view, angiogenesis following ischemic muscle injury in mice was not driven by endothelial sprouting. Instead, real-time imaging revealed the emergence of wide-caliber, primordial conduits with ultralow flow that rapidly transformed into a hierarchical neocirculation by transluminal bridging and intussusception. This process was accelerated by inhibiting vascular endothelial growth factor receptor-2 (VEGFR2). We probed this response by developing the first live-cell model of transluminal endothelial bridging using microfluidics. Endothelial cells subjected to ultralow shear stress could reposition inside the flowing lumen as pillars. Moreover, the low-flow lumen proved to be a privileged location for endothelial cells with reduced VEGFR2 signaling capacity, as VEGFR2 mechanosignals were boosted. These findings redefine regenerative angiogenesis in muscle as an intussusceptive process and uncover a basis for its launch.

The biomechanical and morphological characteristics of the ligamentum mucosum and its potential role in anterior knee pain.

BACKGROUND: The ligamentum mucosum is composed of dense regular connective tissue and traverses from the distal femur to the infrapatellar fat pad. While the gross and histologic morphology has been studied, there is currently no evidence concerning the biomechanical properties of the ligamentum mucosum and the potential of anterior knee pain. The purpose of this study was to determine the anatomical, mechanical and histological properties of the ligamentum mucosum. METHODS: Dissections were performed on cadaveric knee specimens (N = 18) and histological analysis (n = 6) was performed to define the anatomical characteristics of the ligamentum mucosum using standard hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical methods. Biomechanical testing (n = 5) was conducted to determine the tensile properties of the ligamentum mucosum. The peak load at failure, stiffness, and strain were analyzed. RESULTS: Sixty-four percent of the knees had a ligamentum mucosum and the histological analysis confirmed it to be composed of dense regular connective tissue. Small peripheral nerves were identified in the junction between the ligamentum mucosum and the fat pad. The average (SD) peak force of failure, stiffness, and strain were 31.9 N (19.0), 5.1 N/mm (3.59), and 0.83 (0.14), respectively. CONCLUSIONS: The tensile strength and stiffness of the ligamentum mucosum is considerably less than the primary stabilizers of the knee joint. Based on these findings, it is improbable that the ligamentum mucosum has a meaningful effect on the kinematics of the extensor mechanism; perturbations of the tissue and the connected infrapatellar fat pad could potentially play a role in the pathogenesis of anterior knee pain.

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: Bowman's 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.

Regulation of vascular smooth muscle cell phenotype in three-dimensional coculture system by Jagged1-selective Notch3 signaling.

The modulation of vascular smooth muscle cell (VSMC) phenotype is an essential element to fabricate engineered conduits of clinical relevance. In vivo, owing to their close proximity, endothelial cells (ECs) play a role in VSMC phenotype switching. Although considerable progress has been made in vascular tissue engineering, significant knowledge gaps exist on how the contractile VSMC phenotype is induced at the conclusion of the tissue fabrication process. The objectives of this study were as follows: (1) to establish ligand presentation modes on transcriptional activation of VSMC-specific genes, (2) to develop a three-dimensional (3D) coculture model using human coronary artery smooth muscle cells (HCASMCs) and human coronary artery endothelial cells (HCAECs) on porous synthetic scaffolds and, (3) to investigate EC-mediated Notch signaling in 3D cultures and the induction of the HCASMC contractile phenotype. Whereas transcriptional activation of VSMC-specific genes was not induced by presenting soluble Jagged1 and Jagged1 bound to protein G beads, a direct link between HCAEC-bound Jagged1 and HCASMC differentiation genes was observed. Our 3D culture results showed that HCASMCs seeded to scaffolds and cultured for up to 16 days readily attached, infiltrated the scaffold, proliferated, and formed dense confluent layers. HCAECs, seeded on top of an HCASMC layer, formed a distinct, separate monolayer with cell-type partitioning, suggesting that HCAEC growth was contact inhibited. While we observed EC monolayer formation with 200,000 HCAECs/scaffold, seeding 400,000 HCAECs/scaffold revealed the formation of cord-like structures akin to angiogenesis. Western blot analyses showed that 3D coculture induced an upregulation of Notch3 receptor in HCASMCs and its ligand Jagged1 in HCAECs. This was accompanied by a corresponding induction of the contractile HCASMC phenotype as demonstrated by increased expression of smooth muscle-α-actin (SM-α-actin) and calponin. Knockdown of Jagged1 with siRNA showed a reduction in SM-α-actin and calponin in cocultures, identifying a link between Jagged1 and the expression of contractile proteins in 3D cocultures. We therefore conclude that the Notch3 signaling pathway is an important regulator of VSMC phenotype and could be targeted when fabricating engineered vascular tissues.

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.

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.

Three-dimensional topography of synthetic scaffolds induces elastin synthesis by human coronary artery smooth muscle cells.

Due to the important structural and signaling roles of elastin in vascular stability, engineered human vascular tissues must incorporate elastin. However, despite considerable progress toward engineering of elastin-containing vascular tissues from animal cells, currently engineered vascular tissues using human cells largely lack elastin. In this study, we evaluated the effect of scaffold topography (two dimensional [2D] vs. three dimensional [3D]) on elastogenesis in adult human coronary artery smooth muscle cells (HCASMCs). We report that elastin gene expression by HCASMCs was increased by twofold after 4 days of culture in porous 3D polyurethane scaffolds. Transforming growth factor ß1 (TGF-ß1) further increased elastin gene expression in 3D cultures but not in 2D cultures. To evaluate if gene expression is translated into elastin synthesis, both 2D and 3D cultures were analyzed using Western blots. We show that only HCASMCs in 3D scaffolds produced elastin, suggesting that scaffold geometry itself is an important cue for elastogenesis. Moreover, TGF-ß1 enhanced elastin synthesis in 3D, but had no effect on cells grown on 2D surfaces. TGF-ß1, known to induce vascular smooth muscle cells (VSMC) differentiation, upregulated contractile VSMC marker proteins smooth muscle-α-actin and calponin in cells on 2D surfaces. Interestingly, in 3D scaffolds, TGF-ß1 failed to upregulate these differentiation marker proteins for at least 7 days, but did so in cells cultured for 14 days, whereas elastin synthesis was not affected. To our knowledge this study is the first to successfully demonstrate that adult human VSMC can produce elastin when seeded on 3D scaffolds and to directly compare the effect of scaffold topography on elastin synthesis. Knowledge about the conditions required to regulate the phenotype of human VSMCs is paramount to engineer elastin-containing autologous human vascular substitutes.

Smooth muscle alpha-actin and calponin expression and extracellular matrix production of human coronary artery smooth muscle cells in 3D scaffolds.

For a tissue-engineered coronary artery substitute to be a viable clinical option in the treatment of vascular diseases, it is necessary to use tissue-specific human cells. Coronary artery smooth muscle cells are the main resident cells in the tunica media of arteries. In this work, we examined the behavior and differentiation state of human coronary artery smooth muscle cells (HCASMCs) when cultured on 3D polyurethane scaffolds to fabricate hybrid vascular tissues. As the mechanical strength of the scaffold is an important element in engineered hybrid vascular substitutes, porous 3D polyurethane scaffolds fabricated using paraffin spheres and ammonium chloride particles were tested for their mechanical properties both in tension and in compression. The use of ammonium chloride particles as porogen generated scaffolds with superior mechanical properties, which are suitable for vascular tissue engineering. When seeded on uncoated, fibronectin-coated, and Matrigel-coated scaffolds, HCASMCs were well spread and started producing collagen as judged by histochemical analysis but appeared to lack elastin production. Fibronectin coating appeared to promote the infiltration of HCASMCs into the scaffold better than Matrigel coating but did not appear to affect the expression of collagen and elastin. Western blot analyses after successful cell recovery from the scaffolds indicated that HCASMCs, after culturing for 4 and 7 days, expressed similar amounts of smooth muscle alpha-actin and calponin regardless of extracellular matrix coating. Taken together, our data showed that the behavior and differentiation phenotype of HCASMCs can be analyzed after culture in 3D polyurethane scaffolds to establish appropriate conditions that will favor the fabrication of hybrid-engineered vascular substitutes.

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.

Inducible NO synthase (iNOS) in human neutrophils but not pulmonary microvascular endothelial cells (PMVEC) mediates septic protein leak in vitro.

Sepsis-induced acute lung injury (ALI) is characterized by injury of the pulmonary microvascular endothelial cells (PMVEC) leading to high-protein pulmonary edema. Inducible NO synthase (iNOS) mediates trans-PMVEC protein leak in septic mice in vivo and in murine PMVEC under septic conditions in vitro, but the role of iNOS in human PMVEC protein leak has not been addressed. We hypothesized that iNOS in human neutrophils, but not human PMVEC, mediates septic trans-PMVEC protein leak in vitro. We isolated human PMVEC from lung tissue using magnetic bead-bound anti-PECAM antibody and assessed Evans blue albumin leak across human PMVEC monolayers under septic conditions in the presence/absence of human neutrophils. PMVEC were used at passages 3-4, seeded on 3 mum Transwell inserts and grown to confluence. Cytomix-stimulated trans-PMVEC albumin leak was not attenuated by pre-treatment with 1400 W, a selective iNOS inhibitor, or l-NAME, a non-selective NOS inhibitor. In neutrophil-PMVEC co-culture, basal unstimulated trans-EB-albumin leak was 0.6+/-0.3%, which was increased by cytomix stimulation to 11.5+/-4.4%, p<0.01. Cytomix-stimulated EB-albumin leak in neutrophil-PMVEC co-cultures was inhibited by pre-treatment with 1400 W (3.8+/-1.0%, p<0.05) or l-NAME (4.0+/-1.1%, p<0.05). Pre-treatment of neutrophil-PMVEC co-cultures with PEG-SOD (superoxide scavenger) and FeTPPS (peroxynitrite scavenger) also significantly attenuated neutrophil-dependent cytomix-stimulated leak (4.7+/-3.0%, p<0.05; 0.5+/-1.0%, p<0.01, respectively). In conclusion, trans-human PMVEC albumin leak under septic conditions is dependent on iNOS activity specifically in neutrophils, but not in PMVEC themselves. Septic neutrophil-dependent trans-PMVEC albumin leak may be mediated by peroxynitrite.

Polyurethane biomaterials for fabricating 3D porous scaffolds and supporting vascular cells.

Successful tissue engineering of vascular grafts largely depends on synthetic scaffolds that support the survival, proliferation, and differentiation of seeded cells. To investigate the utility of polyurethanes for vascular tissue engineering, three-dimensional porous polyurethane scaffolds with highly interconnected pore structures were fabricated by a pressure differential/particulate leaching technique. Ammonium chloride and paraffin porogens were prepared to fabricate the scaffolds. Grinding of ammonium chloride resulted in particulates with uniform particle sizes but irregular shapes. Paraffin particulates made by a dispersion method, on the other hand, had spherical shapes and uniform particle sizes. Polyurethane scaffolds fabricated from these particulates had open faced, highly interconnected channels that could allow cellular infiltration and nutrient delivery. Human coronary artery smooth muscle and endothelial cell interactions with polyurethane surfaces revealed these biomaterials to maintain the contractile phenotype of human coronary artery smooth muscle cells and the formation of endothelial monolayers. During longer culture times, surface modification with cell adhesive extracellular matrix (ECM) protein promoted vascular cell proliferation, maintenance of the differentiated phenotype and endothelial monolayer integrity. Our results suggest that these polyurethanes, in conjunction with cell adhesive ECM proteins, could also support vascular cells in three-dimensional bioreactor-based culture conditions.

The p110delta isoform of PI3K differentially regulates beta1 and beta2 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. METHODS: The authors employed the PI3K p110delta catalytic subunit specific inhibitor IC87114 (2 microM) and the broad-spectrum PI3K inhibitory agents LY294002 (50 microM) and wortmannin (100 nM), to examine the role of PI3Kdelta in monocyte diapedesis through endothelial monolayers and its role in monocyte adhesion and spreading upon carpets of ICAM-1 or VCAM-1. They further explored the effects of PI3Kdelta inhibition on the activation state of beta1 and beta2 integrins with immunocytochemistry and flow cytometry. RESULTS: In human peripheral blood monocytes IC87114 was as effective as wortmannin and LY294002 at inhibiting diapedesis, however, in THP-1 cells LY294002 and wortmannin caused a 5-fold reduction in diapedesis, while IC87114 only decreased diapedesis 2-fold. PI3Kdelta activity was specifically required for THP-1 cell adhesion and spreading on VCAM-1, but not on ICAM-1 protein substrates. Flow cytometric analysis demonstrated that PI3Kdelta inhibition decreased the amount of conformationally active beta 1-integrins, while having no effect on the prevalence of conformationally active beta 2-integrins expressed on the cell surface. In addition, PI3Kdelta inhibition resulted in a 4-fold decrease in the activation state of Rac-1 and Cdc42. CONCLUSIONS: These results demonstrate the specific necessity of PI3Kdelta in regulating monocytic integrin activation and the general role of PI3K signaling during diapedesis, implicating PI3K as a target for therapeutic intervention.

Elastin biosynthesis: The missing link in tissue-engineered blood vessels.

Nearly 20 years have passed since Weinberg and Bell attempted to make the first tissue-engineered blood vessels. Following this early attempt, vascular tissue engineering has emerged as one of the most promising approaches to fabricate orderly and mechanically competent vascular substitutes. In elastic and muscular arteries, elastin is a critical structural and regulatory matrix protein and plays an important and dominant role by conferring elasticity to the vessel wall. Elastin also regulates vascular smooth muscle cells activity and phenotype. Despite the great promise that tissue-engineered blood vessels have to offer, little research in the last two decades has addressed the importance of elastin incorporation into these vessels. Although cardiovascular tissue engineering has been reviewed in the past, very little attention has been given to elastin. Thus, this review focuses on the recent advances made towards elastogenesis and the challenges we face in the quest for appropriate functional vascular substitutes.

Albumin leak across human pulmonary microvascular vs. umbilical vein endothelial cells under septic conditions.

Human pulmonary microvascular endothelial cell (HPMVEC) injury is central to the pathophysiology of human lung injury. However, septic HPMVEC barrier dysfunction and the contribution of neutrophils have not been directly addressed in vitro. Instead, human EC responses are often extrapolated from studies of human umbilical vein EC (HUVEC). We hypothesized that HUVEC was not a good model for investigating HPMVEC barrier function under septic conditions. HPMVEC was isolated from lung tissue resected from lung cancer patients using magnetic bead-bound anti-PECAM-1 antibody. In confluent monolayers in 3-mum cell-culture inserts, we assessed trans-EC Evans-Blue (EB)-conjugated albumin leak under basal, unstimulated conditions and following stimulation with either lipopolysaccharide or a mixture of equal concentrations of TNF-alpha, IL-1beta and IFN-gamma (cytomix). Basal EB-albumin leak was significantly lower across HPMVEC than HUVEC (0.64 +/- 0.06% vs. 1.13 +/- 0.10%, respectively, P < 0.001). Lipopolysaccharide and cytomix increased leak across both HPMVEC and HUVEC in a dose-dependent manner, with a similar increase relative to basal leak in both cell types. The presence of neutrophils markedly and dose-dependently enhanced cytomix-induced EB-albumin leak across HPMVEC (P < 0.01), but had no effect on EB-albumin leak across HUVEC. Both cytomix and lipopolysaccharide-induced albumin leak was not associated with a loss of cell viability. In conclusion, HPMVEC barrier dysfunction under septic conditions is dramatically enhanced by neutrophil presence, and HUVEC is not a suitable model for studying HPMVEC septic barrier responses. The direct study of HPMVEC septic responses will lead to a better understanding of human lung injury.

Interleukin-1beta 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-1beta exposure of the endothelium. METHODS: Laser scanning confocal microscopy and biochemical analysis were used to determine the effect of IL-1beta pretreatment of the endothelium on adherens junctional morphology and monocyte transcellular diapedesis in cocultures of human peripheral blood monocytes and coronary artery endothelial cells. RESULTS: IL-1beta pretreatment caused a 40% decrease in the number of migrating monocytes that used a transcellular route of diapedesis, and resulted in elongate endothelial cell morphology, a reorganization of the F-actin cytoskeleton, and a significant decrease in transendothelial electrical resistance. In IL-1beta treated monolayers, VE-cadherin and its associated catenins were distributed in a punctate pattern in comparison to the lacy pattern seen in control monolayers. Coimmunoprecipitation of VE-cadherin molecular assemblies revealed that IL-1beta-mediated changes in distribution were associated with a decrease in the presence of cadherin/catenin complexes in the detergent insoluble fraction. CONCLUSIONS: IL-1beta-induced rearrangement of interendothelial adherens junctions facilitates paracellular diapedesis at the expense of transcellular diapedesis.

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

INTRODUCTION: Metastasis 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. METHOD: To 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. RESULTS: HBL100 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. CONCLUSION: Collectively, our results suggest that heterocellular GJIC between breast tumor cells and endothelial cells may be an important regulatory step during metastasis.

Beta3 integrins facilitate matrix interactions during transendothelial migration of PC3 prostate tumor cells.

BACKGROUND: beta3 integrins play a role in metastatic progression of prostate cancer by mediating adhesion of cancer cells to endothelium and migration through extracellular matrix (ECM). However, the role of beta3 integrins during transendothelial migration (TEM) of prostate tumor cells is poorly understood. We examined the role of beta3 integrins in TEM of PC3 human prostate cancer cells through a monolayer of human lung microvascular endothelial cells (HLMVECs). METHODS: PC3 cells were challenged with beta3 integrin antibodies or antisense nucleotides and their efficiency to migrate through monolayers of endothelial cells (ECs) was assessed using confocal microscopy. RESULTS: beta3 integrins in PC3 cells are not localized in focal contacts and their blockade significantly inhibited TEM by over 50% preferentially during late stages of migration. Formation of PC3 cell pseudopodia on matrigel was significantly reduced by beta3 integrin antisense oligonucleotides. CONCLUSIONS: beta3 integrins play important roles during TEM of PC3 cells while interacting with the matrix underneath the endothelium. These interactions are independent of the ability to cluster beta3 integrins into focal adhesions.

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. METHODS: Human umbilical vein endothelial cells (HUVECs) were grown to confluence on porous membranes and activated with interleukin-1beta, and PMN transendothelial migration was facilitated by formyl-methionyl-leucyl-phenylalanine. Using dual immunofluorescence staining and laser scanning confocal microscopy, we assessed the effects of PMN-endothelial cell adhesive interactions (i.e., adhesion to and emigration across monolayers) on the AJ components vascular endothelial (VE)-cadherin, beta-catenin, alpha-catenin, and gamma-catenin. RESULTS: In the AJ immediately adjacent to the adherent PMN, there was a loss of staining for some of the AJ components. AJ components further away from HUVEC-PMN adhesive interactions were unaffected. An iterative approach indicated that the four components were sequentially lost from the AJ. beta-catenin was lost first, followed by VE-cadherin, alpha-catenin, and, finally, gamma-catenin. In the absence of PMNs, the cross-linking of VE-cadherin, but not platelet endothelial cell adhesion molecule-1 or intercellular adhesion molecule-1, increased the cytoplasmic accumulation of beta-catenin. During PMN transendothelial migration, all of the junctional components under study were lost at the immediate site of monolayer penetration. Again, at regions removed from the actual site of PMN penetration of the monolayers, the AJ components were unaffected. PMN-induced disorganization of the AJs was partially prevented by an elastase inhibitor. CONCLUSIONS: These findings suggest that adherent PMNs induce a localized, sequential disassembly of AJs, which is partially mediated by PMN-derived elastase and involves the initial loss of an intracellular component of AJs (i.e., beta-catenin).

Endothelin-a receptor in rat skeletal muscle microvasculature.

Although the effect of endothelin-1 (ET-1) on vascular tone has been studied extensively at the arterial/arteriolar level, little is known about the direct effect of ET-1 at the level of the capillary. Using intravital microscopy, we determined capillary red blood cell velocity and arteriolar diameter responses to ET-1, ET(A)-receptor blocker BQ-123, and ET(B)-receptor blocker BQ-788 applied locally on capillaries in rat extensor digitorum longus (EDL) muscle. Using immunohistochemistry, we examined capillaries in this muscle and microvascular endothelial cells isolated from this muscle for immunoreactivity with ET(A)-receptor antibody. ET-1 (10(-9) to 10(-5) M in micropipette) caused quick reductions (i.e., within several seconds), whereas BQ-123 (10(-8) to 10(-4) M) and BQ-788 (10(-6) and 10(-4) M) caused quick increases, in both velocity and diameter. Capillaries and endothelial cells showed ET(A)-receptor immunoreactivity. We conclude that the microvasculature of the rat EDL muscle is sensitive to ET-1 and its receptor blockers and that the ET(A) receptor may be present in the capillary wall of this muscle, including the endothelium.