Comparative study of adhesion molecule expression in cultured human macro- and microvascular endothelial cells

Culture systems as models for disease are only valid as long as they are comparable to in vivo conditions. The phenotype of cultured endothelial cells (ECs) has only been sporadically compared to the corresponding phenotype in vivo. Thus, we compared by immunolocalization the endothelial expression of ICAM-1, VCAM, and E-selectin in vivo in stimulated/unstimulated human umbilical vein endothelial cells (HUVEC) as a model for macrovascular ECs and stimulated/unstimulated HPMEC (human pulmonary microvessel endothelial cells) as a model for pulmonary microvascular ECs with that in human lungs in vivo (normal and ARDS). Proinflammatory stimuli in vitro were used to stimulate conditions relevant for ARDS. ICAM-1 expression in stimulated HUVEC/HPMEC correlated well with in vivo expression (macro- and microvessels). For E-selectin, the staining pattern in macro/microvessels correlated moderately with unstimulated and well with stimulated HUVEC/HPMEC. For VCAM a good correlation was found for stimulated/unstimulated HUVEC and unstimulated HPMEC. The expression patterns in stimulated HUVEC corresponded well for all three molecules with those in vivo. Thus, the expression patterns in vitro are only partially transferable to in vivo conditions. The study suggests that E-selectin- and VCAM-coated beads could potentially serve in the isolation process of arteriolar and venular ECs.     

Differential adhesion of polymorphous neutrophilic granulocytes to macro- and microvascular endothelial cells under flow conditions.

OBJECTIVE: As one of the important active barriers in the human organism, endothelial cells (EC) play a central role in the biological reaction to a variety of stimuli, e.g. during the induction and regulation of inflammation, as well as in the reaction to transplantation and biomaterial implantation. In the study of endothelial function, the most widely used in vitro model is that of human umbilical vein EC (HUVEC), i.e. an EC type of embryonic and macrovascular origin. However, many of the important pathological processes occur at microvascular level, thus questioning the validity of the HUVEC model. Moreover, the morphological and functional heterogeneity of the endothelium in the various organs, e.g. kidney, liver and lung, must be taken into consideration. The purpose of the present study was to use a dynamic cell culture system to compare the reactions of HUVEC and human pulmonary microvascular EC (HPMEC) to pro-inflammatory stimulation. METHODS: HUVEC and HPMEC in monolayer culture were stimulated by tumor necrosis factor-alpha (TNFalpha) in a parallel-plate flow chamber. Short- (4 h) and long-term (12 h) stimulation were compared. As a functional parameter, the adhesion of human peripheral blood polymorphonuclear granulocytes (PMN) to EC was quantitated both under venous and arterial flow conditions. RESULTS: Short-term (4 h) TNFalpha stimulation and venous flow conditions elicited a 32% higher PMN adhesion to HPMEC compared with HUVEC, whereas under arterial flow conditions no statistically significant differences were found. Following longer-term (12 h) TNFalpha stimulation, PMN adhesion to HPMEC was 65% higher than to HUVEC under venous flow. Under arterial flow no differences were detected. CONCLUSION: The present results provide new data on the heterogeneity of the endothelium and affect a central element in microvascular pathology, namely granulocyte-endothelial interactions. Moreover, this paper emphasizes the necessity to evaluate the in vitro models of the endothelium with respect to the extrapolation to the situation in vivo.     

Generation of human pulmonary microvascular endothelial cell lines.

The limited lifespan of human microvascular endothelial cells in cell culture represents a major obstacle for the study of microvascular pathobiology. To date, no endothelial cell line is available that demonstrates all of the fundamental characteristics of microvascular endothelial cells. We have generated endothelial cell lines from human pulmonary microvascular endothelial cells (HPMEC) isolated from adult donors. HPMEC were cotransfected with a plasmid encoding the catalytic component of telomerase (hTERT) and a plasmid encoding the simian virus 40 (SV40) large T antigen. Cells transfected with either plasmid alone had an extended lifespan, but the cultures eventually entered crisis after several months of proliferation. Only those cells that were transfected with both plasmids acquired the capacity to grow in vitro without demonstrating major crisis, and these cells have been in culture for 24 months. HPMEC isolated from two different donors were used, generating two populations of immortalized cells, HPMEC-ST1 and HPMEC-ST2. Single cell-derived clones of the immortalized cells HPMEC-ST1 exhibited growth characteristics that were similar to those of the parental HPMEC. One selected clone, HPMEC-ST1.6R, displayed all major constitutively expressed and inducible endothelial phenotypic markers, including platelet endothelial cell adhesion molecule (PECAM-1, CD31), von Willebrand factor (vWF), and the adhesion molecules, intercellular adhesion molecule (ICAM-1), vascular adhesion molecule (VCAM-1), and E-selectin. In addition, an angiogenic response was demonstrated by sprout formation on a biological extracellular matrix (Matrigel). The HPMEC-ST1.6R cells did not form tumors in nude mice. The microvascular endothelial cell line, HPMEC-ST1.6R, will be a valuable tool for the study of microvascular endothelial physiology and pathology including gene expression, angiogenesis, and tumorigenesis.     

Interleukin-11 and interleukin-6 protect cultured human endothelial cells from H2O2-induced cell death

Acute lung injury is a frequent and treatment-limiting consequence of therapy with 100% oxygen. Previous studies have determined that both interleukin (IL)-6 and IL-11 are protective in oxygen toxicity. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals, and both IL-6 and IL-11 markedly diminished this cell death response. However, the mechanism(s) by which these cytokines protect cells from death is unclear. In the present study, we characterized the effects of H2O2 on subconfluent human umbilical vein endothelial cell (HUVEC) and human pulmonary microvascular endothelial cell (HPMEC) cultures. We found that preincubation of HUVEC cultures with either IL-6 or IL-11 diminished H2O2 (1.0 mM)-induced cell death. Similar effects were noted with HPMEC showing that this effect is not HUVEC-specific. The protective effects of both IL-6 and IL-11 were not associated with any changes in antioxidants and were decreased by approximately 80% in the presence of U0126, a specific inhibitor of MEK-1-dependent pathways. The cytoprotective effects of IL-11 and IL-6 were also completely eliminated in STAT3 dominant-negative transduced HUVEC cultures. These studies demonstrate that IL-6 and IL-11 both confer cytoprotective effects that diminish oxidant-mediated endothelial cell injury. They also demonstrate that this protection is mediated, at least in part, by a STAT3 and MEK-1-dependent specific signal transduction pathway(s).     

Response of micro- and macrovascular endothelial cells to starch-based fiber meshes for bone tissue engineering

The establishment of a functional vasculature is as yet an unrealized milestone in bone reconstruction therapy. For this study, fiber-mesh scaffolds obtained from a blend of starch and poly(caprolactone) (SPCL), that have previously been shown to be an excellent material for the proliferation and differentiation of bone marrow cells and thereby represent great potential as constructs for bone regeneration, were examined for endothelial cell (EC) compatibility. To be successfully applied in vivo, this tissue engineered construct should also be able to support the growth of ECs in order to facilitate vascularization and therefore assure the viability of the construct upon implantation. The main goal of this study was to examine the interactions between ECs and SPCL fiber meshes. Primary cultures of HUVEC cells were selected as a model of macrovascular cells and the cell line HPMEC-ST1.6R as a model for microvascular ECs. Both macro- and microvascular ECs adhered to SPCL fiber-mesh scaffolds and grew to cover much of the available surface area of the scaffold. In addition, ECs growing on the SPCL fibers exhibited a typical morphology, maintained important functional properties, such as the expression of the intercellular junction proteins, PECAM-1 and VE-cadherin, the expression of the most typical endothelial marker vWF and sensitivity to pro-inflammatory stimuli, as shown by induction of the expression of cell adhesion molecules (CAMs) by lipopolysaccharide (LPS). These data indicate that ECs growing on SPCL fiber-mesh scaffolds maintain a normal expression of EC-specific genes/proteins, indicating a cell compatibility and potential suitability of these scaffolds for the vascularization process in bone tissue engineering in vivo.     

Defining optimum conditions for the ex vivo expansion of human umbilical cord blood cells. Influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels

Ex vivo expansion of human umbilical cord blood cells (HUCBC) is explored by several investigators to enhance the repopulating potential of HUCBC. We performed experiments using either Ficoll-separated or CD34+-selected HUCBC from the same donation in serum-free medium. CD34-purified HUCBC were cultured on either human umbilical vein endothelial cells (HUVEC) or irradiated bone marrow-derived stroma cells (BMSC) with addition of different cytokines. In addition, we tested the expansion of HUCBC in culture vessels with continuous rotation. CD34 enrichment led to a significant increase in the expansion factor of CD34+ cells compared with unmanipulated HUCBC. BMSC were more efficient in amplifying early progenitors than HUVEC. Optimum results were reached by a combination of SCF, FLT-3L at 300 ng/ml and IL-3 at 50 ng/ml. No significant improvement in the expansion of CD34+/38- primitive progenitors could be obtained with other combinations. Addition of megakaryocyte-derived growth and development factor to each growth factor cocktail improved the expansion results. Continuous rotation of culture vessels did not ameliorate the expansion rate of the analyzed subsets. Culture conditions separating stroma and HUCBC by a semipermeable membrane improved the expansion factors of CD34+, CD34+/38-, and CD34+/41+ cells and CFU-GM compared with contact cultures. These data might be useful when designing culture systems for clinical scale ex vivo expansion of HUCBC.     

Platelet-endothelial cell adhesion molecule-1 (CD31) redistributes from the endothelial junction and is not required for the transendothelial migration of melanoma cells

We have examined the role of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) during the transendothelial migration of melanoma cells using a novel in vitro system. Comparable studies have suggested the involvement of PECAM-1 in leukocyte transendothelial migration. Such studies have been confirmed using in vivo models of inflammation. These studies prompted us to examine the role of PECAM-1 in tumor cell transendothelial migration. Anti-PECAM-1 monoclonal antibodies, known to block leukocyte transendothelial migration, were tested in co-cultures of human melanoma cells seeded on a monolayer of human lung microvascular endothelial cells. None of these antibodies inhibited the transmigration of melanoma cells. Moreover, confocal microscopy revealed the dissolution of the PECAM-1 adhesion complexes in the endothelial junctions associated with melanoma cells and the lack of PECAM-1 in heterotypic contacts between transmigrating melanoma cells and adjacent endothelial cells. These data, therefore, indicate that PECAM-1 is not required for the transendothelial migration of melanoma cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

An improved endothelial barrier model to investigate dengue haemorrhagic fever

A cell culture model suitable for studies of dengue haemorrhagic fever was developed, based on culture of primary human umbilical vein endothelial cells (HUVECs) on a permeable membrane. By electron microscopy, cultured HUVECs at day 11 resembled morphologically microvascular endothelium. Endothelial barrier function was assessed by measuring transendothelial flux of albumin. Instead of using a labelled tracer molecule, an enzyme-linked immunosorbent assay (ELISA) was developed to measure concentrations of native human albumin. The permeability characteristics of the HUVEC monolayer were found to be improved significantly (approximately 1 log reduction in permeability coefficient for albumin) by culturing HUVECs in human serum rather than fetal calf serum. Permeability coefficients for albumin in the range 1-4 x 10(-7) cm/s were achieved, which is an improvement on previous in vitro models of the endothelial barrier. Comparison of transendothelial flux of albumin and urea provided evidence of molecular sieving by the HUVEC monolayer. Moreover, tumour necrosis factor-alpha induced a dose-dependent, reversible increase in permeability of the HUVEC monolayer. This endothelial barrier model thus has many important characteristics that resembled human microvascular endothelium and is an improvement on the previous model proposed for studies of dengue haemorrhagic fever.     

Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro

We have established a coculture system of human distal lung epithelial cells and human microvascular endothelial cells in order to study the cellular interactions of epithelium and endothelium at the alveolocapillary barrier in both pathogenesis and recovery from acute lung injury. The aim was to determine conditions for the development of functional cellular junctions and the formation of a tight epithelial barrier similar to that observed in vivo. The in vitro coculture system consisted of monolayers of human lung epithelial cell lines (A549 or NCI H441) and primary human pulmonary microvascular endothelial cells (HPMEC) on opposite sides of a permeable filter membrane. A549 failed to show sufficient differentiation with respect to formation of a tight epithelial barrier with intact cell-cell junctions. Stimulated with dexamethasone, the cocultures of NCI H441 and HPMEC established contact-inhibited differentiated monolayers, with NCI H441 showing a continuous, circumferential immunostaining of the tight junctional protein, ZO-1 and the adherens junction protein, E-cadherin. The generation of a polarized epithelial cell monolayer with typical junctional structures was confirmed by transmission electron microscopy. Dexamethasone treatment resulted in average transbilayer electrical resistance (TER) values of 500 Omega cm(2) after 10-12 days of cocultivation and correlated with a reduced flux of the hydrophilic permeability marker, sodium-fluorescein. In addition, basolateral distribution of the proinflammatory cytokine tumour necrosis factor-alpha caused a significant reduction of TER-values after 24 h exposure. This decrease in TER could be re-established to control level by removal of the cytokine within 24 h. Thus, the coculture system of the NCI H441 with HPMEC should be a suitable in vitro model system to examine epithelial and endothelial interactions in the pathogenesis of acute lung injury, infectious lung diseases and toxic lung injury. In addition, it could be used to improve techniques of lung drug delivery that also requires a functional barrier.     

免疫球蛋白超家族黏附分子在淋巴管和不同血管内皮细胞的表达

目的比较免疫球蛋白超家族黏附分子在淋巴管、大血管和微血管内皮细胞的表达特点,探讨免疫球蛋白超家族黏附分子在淋巴管内皮细胞表达的意义。方法从狗的胸导管、颈总动脉、颈内静脉、肺微血管分离内皮细胞,利用免疫荧光标记法检测PECAM-1、ICAM-1、ICAM-3、VCAM-1和CD44在各种内皮细胞的表达,在荧光显微镜和激光共聚焦扫描显微镜下观察,并用图像分析仪分析表达强度。结果动脉、静脉和肺微血管内皮细胞表达PECAM—1、ICAM—1、ICAM-3、VCAM—1和CD44。其中,ICAM-1和ICAM-3的表达较弱。VCAM—1在动脉和肺微血管内皮细胞的表达比静脉强。淋巴管内皮细胞表达PECAM—1、ICAM—1、ICAM-3和CD44,未观察到VCAM—1的表达。ICAM-3和CD44的表达比血管内皮细胞强。结论与动脉、静脉和微血管内皮细胞比较,淋巴管内皮细胞不表达VCAM—1,而ICAM-3和CD44表达较强,这有助于解释淋巴细胞和肿瘤细胞与淋巴管内皮的黏附以及淋巴管新生的机制。     

PECAM-1 affects GSK-3beta-mediated beta-catenin phosphorylation and degradation

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) regulates a variety of endothelial and immune cell biological responses. PECAM-1-null mice exhibit prolonged and increased permeability after inflammatory insults. We observed that in PECAM-1-null endothelial cells (ECs), beta-catenin remained tyrosine phosphorylated, coinciding with a sustained increase in permeability. Src homology 2 domain containing phosphatase 2 (SHP-2) association with beta-catenin was diminished in PECAM-1-null ECs, suggesting that lack of PECAM-1 inhibits the ability of this adherens junction component to become dephosphorylated, promoting a sustained increase in permeability. beta-Catenin/Glycogen synthase kinase 3 (GSK-3beta) association and beta-catenin serine phosphorylation levels were increased and beta-catenin expression levels were reduced in PECAM-1-null ECs. Glycogen synthase kinase 3 (GSK-3beta) serine phosphorylation (inactivation) was blunted in PECAM-1-null ECs after histamine treatment or shear stress. Our data suggest that PECAM-1 serves as a critical dynamic regulator of endothelial barrier permeability. On stimulation by a vasoactive substance or shear stress, PECAM-1 became tyrosine phosphorylated, enabling recruitment of SHP-2 and tyrosine-phosphorylated beta-catenin to its cytoplasmic domain, facilitating dephosphorylation of beta-catenin, and allowing reconstitution of adherens junctions. In addition, PECAM-1 modulated the levels of beta-catenin by regulating the activity of GSK-3beta, which in turn affected the serine phosphorylation of beta-catenin and its proteosomal degradation, affecting the ability of the cell to reform adherens junctions in a timely fashion.     

Inhibition of pre-B cell colony-enhancing factor attenuates inflammation and apoptosis induced by pandemic H1N1 2009 in lung endothelium

The recent pandemic influenza A (H1N1 2009) virus infection has caused acute lung injury in susceptible population resulting in high mortality in ICU patients. In this report, we observed the effect of pre-B cell colony-enhancing factor (PBEF) on the inflammation and apoptosis in H1N1-infected human pulmonary microvascular endothelial cells (HPMECs). We constructed an in vitro HPMEC monolayer model. The results showed that H1N1 2009 induced the increased expression of inflammatory cytokines (IL-6/IL-8/TNF-α/IP-10) and apoptosis factors (FasL/TRAIL) in infected HPMECs. However, PBEF silencing with siRNA inhibited the expression of some inflammatory cytokines and decreased the apoptosis mediated by FasL. We conclude that PBEF might be partially responsible for the localized inflammatory response to H1N1 2009 in the lung microvascular endothelium and the H1N1-induced endothelial cell apoptosis probably through the FasL-mediated pathway.     

Overexpression of stromal cell-derived factor-1 enhances endothelium-supported transmigration, maintenance, and proliferation of hematopoietic progenitor cells

To clarify the direct effects of aberrant overexpression of stromal cell-derived factor-1 (SDF-1) by the human endothelium on circulating progenitor cells, we overexpressed the SDF-1 gene in human umbilical vein endothelial cells using an adenoviral vector (HUVEC/AdeSDF-1) and examined the endothelium-supported trafficking and growth of hematopoietic progenitor cells (HPCs) in mobilized peripheral blood (mPB). In culture, the HUVEC/AdeSDF-1 monolayers induced the migration of mPB CD34(+) cells underneath the endothelium within a few hours, whereas HUVEC monolayers that expressed the LacZ gene (HUVEC/AdeLacZ) did not have this effect. In the Transwell system, the HUVEC/AdeSDF-1 cells supported a higher level of spontaneous transmigration of mPB CD34(+) cells than did the HUVEC/AdeLacZ cells. The co-culturing of mPB CD34(+) cells with HUVEC/ AdeSDF-1 cells led to a greater expansion of CD45(+) cells and colony-forming cells and reduced cellular apoptosis. Furthermore, the co-culturing of mPB CD34(+) cells with HUVEC/AdeSDF-1 cells led to the formation of numerous cobblestone-like areas, whereas co-cultures of mPB CD34(+) cells and HUVEC/AdeLacZ supported only a few cobblestone-like areas. These results indicate that SDF- 1 produced by endothelial cells plays an important role not only in the transmigration but also in the growth of HPCs that are in contact with endothelial cells. Our findings suggest that the enhanced expression and production of SDF-1 in the endothelium are essential steps for stem cell or progenitor cell recruitment to specific tissues and for the maintenance of these cells in situ.     

Adherent cells generated during long-term culture of human umbilical cord blood CD34+ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study, UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin, flt3 ligand, and granulocyte-colony stimulating factor. By week 4-5, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor, human vascular cell adhesion molecule-1, human intracellular adhesion molecule-1, human CD31, E-selectin, and human macrophage. Furthermore, when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer, better expansion of total nucleated cells, CD34(+) cells, and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells, which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells, we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method, one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors, establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering.     

Involvement of endothelial PECAM-1/CD31 in angiogenesis.

The adhesive interactions of endothelial cells with each other and the adhesion receptors that mediate these interactions are probably of fundamental importance to the process of angiogenesis. We therefore studied the effect of inhibiting the function of the endothelial cell-cell adhesion molecule, PECAM-1/ CD31, in rat and murine models of angiogenesis. A polyclonal antibody to human PECAM-1, which cross-reacts with rat PECAM-1, was found to block in vitro tube formation by rat capillary endothelial cells and cytokine-induced rat corneal neovascularization. In mice, two monoclonal antibodies against murine PECAM-1 prevented vessel growth into subcutaneously implanted gels supplemented with basic fibroblast growth factor (bFGF). Taken together these findings provide evidence that PECAM-1 is involved in angiogenesis and suggest that the interactions of endothelial cell-cell adhesion molecules are important in the formation of new vessels.     

CD 4-positive lymphoid cells rescue HIV-1 replication from abortively infected human primary endothelial cells

Summary Human primary endothelial cell cultures, derived from umbilical vein (HUVEC), can be infected by different strains of HIV-1, but mature virus production remains undetectable both in supernatants and in cellular extracts. Yet viral DNA is transiently detectable during the first days of infection, but progressively declines during the subsequent days. This finding is characteristic of abortive infections. Co-culture of HUVEC carrying HIV DNA with activated peripheral blood mononuclear cells or with CD 4-positive lymphoid cells elicited a massive cpe (syncytia formation and cell degeneration) in the latter cells, caused by the establishment of productive HIV-1 infection. HUVEC infected in the presence of AZT were significantly impaired in the ability to transmit the infection of CD 4-positive cells, indicating that active DNA synthesis is required in HUVEC before rescue by CD 4-positive cells.These results are of interest in view of the possibility that endothelial cells can play a role in the transmission of HIV-1 infection from infected pregnant women to the foetuses, and, more generally, suggest a potential role of endothelial cells as a transient reservoir of HIV-1.     

Steady unidirectional laminar flow inhibits monolayer formation by human and rat microvascular endothelial cells.

Endothelialization of artificial vascular grafts is rapid and complete in numerous animal models, including dogs and rats, but not in human patients. One possible explanation for this well-known, yet puzzling observation might be that monolayer formation of human endothelial cells (ECs), and of canine or rodent ECs, is affected differently by flow-induced shear stress. To begin testing this hypothesis, the authors wounded confluent monolayers of cultured rat and human ECs and exposed these cultures for 20 h to unidirectional steady laminar shear stress of 10 dyn/cm(2) induced by fluid flow perpendicular to the wound boundaries. In comparison to experimental control cultures simultaneously maintained under static (no-flow) conditions, flow-induced shear stress attenuated the monolayer formation (sheet migration) in both human and rat ECs. In brief, compared to control, the average human EC monolayer formation under shear was reduced by 33% whereas the average rat EC monolayer formation was reduced by 34%. Furthermore, the cell responses showed a dependence on fluid flow direction that differed per species. When exposed to shear stress, human EC monolayer formation was reduced by 16% in the upstream direction (opposing the direction of flow) and reduced by 50% in the downstream direction (with the direction of flow), whereas rat EC monolayer formation was reduced by 64% upstream and showed no change downstream. These findings suggest that although overall monolayer formation is inhibited by fluid-induced shear stress to the same extent in both species, there are cell type- and/or species-dependent migration responses to fluid-induced shear stress, and that different flow conditions possibly contribute to species-specific patterns of endothelialization.     

Vascularization and gene regulation of human endothelial cells growing on porous polyethersulfone (PES) hollow fiber membranes

Open-cell hollow fibers made of polyethersulfone (PES) manufactured in the absence of solvents with pore diameters smaller than 100 microm were examined for vascularization by human endothelial cells. The goal of this study was to determine whether the 3-D porous character of the PES surface affected human endothelial cell morphology and functions. Freshly isolated human endothelial cells from the skin (HDMEC), from the lung (HPMEC) and from umbilical cords (HUVEC) and two human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS.c1 were added to PES fibers and cell adherence and growth was followed by confocal laser scanning microscopy. Prior coating of PES with gelatin or fibronectin was necessary for adhesion and spreading of cells over the uneven porous surface with time. Confluent cells exhibited typical strong PECAM-1 expression at cell-cell borders. Little expression of the activation markers E-selectin, ICAM-1, and VCAM-1 was observed by RT-PCR of endothelial cells growing on PES. However, after stimulation for 4h by LPS, activation of these markers was observed and it was shown by immunofluorescent staining that induction occurred in most of the cells, thus confirming an intact functionality. Finally, cells growing as a monolayer on PES migrated to form microvessel-like structures when placed under conditions that stimulated angiogenesis. Thus, human endothelial cells grown on fibronectin-coated PES fibers retain important endothelial-cell specific morphological and functional properties and PES may serve as a useful biomaterial in tissue engineering and biotechnology applications.     

The effect of transendothelial migration on eosinophil function

In bronchial asthma, eosinophils found in the airways have an enhanced inflammatory capacity. We hypothesized that, at least in part, changes in functional phenotype are due to the effect of transendothelial migration. To model in vivo eosinophil trafficking to the lung, we cultured human pulmonary microvascular endothelial cell (HPMEC) monolayers on Transwell filters. The HPMECs were activated with interleukin (IL)-1beta to increase cell expression of intercellular adhesion molecule (ICAM)-1 and, hence, eosinophil transmigration. Peripheral blood eosinophils from allergic patients were added to HPMEC-covered Transwell filters and incubated for 3 h at 37 degrees C. The eosinophils were collected from below (migrated cells) and above (nonmigrated cells) the HPMEC monolayer to determine surface receptor expression, in vitro survival, and oxidative burst. Eosinophils never exposed to HPMECs were used as controls. Eosinophil cell surface expression of CD69, human leukocyte-associated antigen-DR (HLA-DR), and CD54 (ICAM-1) was significantly increased after transendothelial migration through IL-1beta-treated HPMECs compared with control cells (CD69: P<0.0005; HLA-DR and CD54: P<0.05) and nonmigrated eosinophils (CD69 and HLA-DR: P<0.05). Moreover, the percent in vitro survival (48 h) of migrated eosinophils was also significantly greater (P<0.0001 by trypan blue exclusion, P< 0.05 by flow cytometry) than that of control or nonmigrated eosinophils. Prolonged survival of migrated eosinophils was inhibited by addition of anti-granulocyte macrophage colony-stimulating factor (GM-CSF) antibodies (P<0.05) to the 48-h survival culture, suggesting that autocrine production of GM-CSF was, at least partially, responsible for increased eosinophil survival. Although GM-CSF protein was not measurable in survival culture supernates, GM-CSF messenger RNA (mRNA) was expressed in both nonmigrated and migrated eosinophils but not in control cells. Similarly, the eosinophils' oxidative burst induced by platelet-activating factor, formylmethionyl leucylphenylalanine, or phorbol myristate acetate was equally, and significantly, increased in both nonmigrated and migrated eosinophils (P<0.05 versus control). Therefore, whereas exposure of eosinophils to cytokine-activated HPMECs can increase surface receptor expression, in vitro survival, GM-CSF mRNA, and the respiratory burst, transendothelial migration can further potentiate receptor expression and survival in migrated cells. These results suggest that the process of transendothelial migration selectively participates in determining the eventual phenotype of airway eosinophils.