Formation Of Endothelial Cell Monolayer Research Articles

Basement Membrane of Tissue Engineered Extracellular Matrix Scaffolds Modulates Rapid Human Endothelial Cell Recellularization and Promote Quiescent Behavior After Monolayer Formation.

Off-the-shelf small diameter vascular grafts are an attractive alternative to eliminate the shortcomings of autologous tissues for vascular grafting. Bovine saphenous vein (SV) extracellular matrix (ECM) scaffolds are potentially ideal small diameter vascular grafts, due to their inherent architecture and signaling molecules capable of driving repopulating cell behavior and regeneration. However, harnessing this potential is predicated on the ability of the scaffold generation technique to maintain the delicate structure, composition, and associated functions of native vascular ECM. Previous de-cellularization methods have been uniformly demonstrated to disrupt the delicate basement membrane components of native vascular ECM. The antigen removal (AR) tissue processing method utilizes the protein chemistry principle of differential solubility to achieve a step-wise removal of antigens with similar physiochemical properties. Briefly, the cellular components of SV are permeabilized and the actomyosin crossbridges are relaxed, followed by lipophilic antigen removal, sarcomeric disassembly, hydrophilic antigen removal, nuclease digestion, and washout. Here, we demonstrate that bovine SV ECM scaffolds generated using the novel AR approach results in the retention of native basement membrane protein structure, composition (e.g., Collagen IV and laminin), and associated cell modulatory function. Presence of basement membrane proteins in AR vascular ECM scaffolds increases the rate of endothelial cell monolayer formation by enhancing cell migration and proliferation. Following monolayer formation, basement membrane proteins promote appropriate formation of adherence junction and apicobasal polarization, increasing the secretion of nitric oxide, and driving repopulating endothelial cells toward a quiescent phenotype. We conclude that the presence of an intact native vascular basement membrane in the AR SV ECM scaffolds modulates human endothelial cell quiescent monolayer formation which is essential for vessel homeostasis.

Nitrate-Functionalized poly(ε-Caprolactone) Small-Diameter Vascular Grafts Enhance Vascular Regeneration via Sustained Release of Nitric Oxide.

Artificial small-diameter vascular grafts (SDVG) fabricated from synthetic biodegradable polymers, such as poly(ε-caprolactone) (PCL), exhibit beneficial mechanical properties but are often faced with issues impacting their long-term graft success. Nitric oxide (NO) is an important physiological gasotransmitter with multiple roles in orchestrating vascular tissue function and regeneration. We fabricated a functional vascular graft by electrospinning of nitrate-functionalized poly(ε-caprolactone) that could release NO in a sustained manner via stepwise biotransformation in vivo. Nitrate-functionalized SDVG (PCL/NO) maintained patency following abdominal arterial replacement in rats. PCL/NO promoted cell infiltration at 3-months post-transplantation. In contrast, unmodified PCL SDVG showed slow cell in-growth and increased incidence of neointima formation. PCL/NO demonstrated improved endothelial cell (EC) alignment and luminal coverage, and more defined vascular smooth muscle cell (VSMC) layer, compared to unmodified PCL SDVG. In addition, release of NO stimulated Sca-1+ vascular progenitor cells (VPCs) to differentiate and contribute to rapid luminal endothelialization. Furthermore, PCL/NO inhibited the differentiation of VPCs into osteopontin-positive cells, thereby preventing vascular calcification. Overall, PCL/NO demonstrated enhanced cell ingrowth, EC monolayer formation and VSMC layer regeneration; whilst inhibiting calcified plaque formation. Our results suggested that PCL/NO could serve as promising candidates for improved and long-term success of SDVG implants.

Endothelial cell monolayer formation on a small-diameter vascular graft surface under pulsatile flow conditions

Objective: to create a cell-populated small-diameter vascular graft (SDVG) using autologous endothelial cells and extracellular matrix proteins, and to evaluate the efficiency of endothelial cell monolayer formation during shear stress preconditioning in a SDVG.Materials and methods. PHBV/PCL tubular scaffolds of vascular grafts were made by electrospinning from a mixture of polyhydroxybutyrate-valerate (PHBV) copolymer and polycaprolactone (PCL) and modified with fibrin. To populate the graft, an endothelial cell culture was isolated from the blood of patients with coronary heart disease. Phenotyping of endothelial colony-forming cell (ECFC) culture was performed by flow cytometry and immunofluorescence microscopy. Cell proliferative and angiogenic activity were also studied. Cell-populated vascular scaffolds were cultured in a pulsatile flow setup with a final shear stress of 2.85 dyne/cm2. The effect of pulsatile flow on monolayer formation was assessed by immunofluorescence, scanning electron microscopy, atomic force microscopy, and whole-transcriptome RNA sequencing.Results. Under the influence of pulsatile flow, endothelial cells that were seeded into the tubular scaffold showed an increase in the expression level of endothelial profile proteins, focal adhesion and cytoskeleton. In contrast to endothelial cell culture on a vascular graft surface under static conditions, when cultured under pulsatile flow with 2.85 dyne/ cm2 shear stress, endothelial lining cells have an increased ability to adhere and are oriented along the pulsatile flow path. Whole-transcriptome RNA sequencing showed that induced shear stress increased expression levels of differentially expressed genes encoding proteins that ensure vascular development, endothelial integrity, and endothelial metabolism. A protocol for fabrication of a personalized cell-populated biodegradable SDVG under pulsatile flow conditions was developed.Conclusion. The use of autologous fibrin and ECFC culture, as well as shear stress preconditioning, allow to obtain a personalized cell-populated SDVG with continuous functional endothelial monolayer adapted to the flow.

Hemocompatibility of Bioprosthetic valve in bovine pericardium is based on fibrin formation and its endothelization

Bioprosthetic heart valves (BV) made of xenogenic pericardium fixed with glutaraldehyde have been developed more than 30 years ago. The concept of cross linking collagenous fibers to ensure in-vivo stability of the scaffold and reducing antigenic immune response by fixation resulted in successful clinical evaluations and a lower risk of thromboembolism thus eliminating the need for a lifelong anticoagulation. To explain these clinical results, an acquired hemocompatibility process has been proposed but never extensively studied because of the impossibility to access not degenerated material. To study and understand mechanisms of acquired hemocompatibility, we explored non degenerated BV from 9 different patients implanted with a Carmat total artificial heart (C-TAH) and four BV duration from 45 to 270 days. We performed classic histopathology and immunophenotypic characterization of explanted BV. Biological deposit has been also explored by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). In short implantation duration time, we observed a fibrin deposit with inflammatory cells and pseudotubes formation. Histological sections showed superficial infiltration of cells, persisting in long duration but not damaging integrity of the pericardium. The fibrin deposit is then organized in a compacted smooth fibrin network in longer implantation durations. Endothelial cells (EC) monolayer covering has also been observed on this fibrin cap, the percentage of the BV surface with EC covering increasing along months. This endothelial cells covering is positive for VE-Cadherin, with flat nucleus and tight junctions. Healthy BV implanted with C-TAH enabled us to study, for the first time, non-degenerated implanted valve in human. Biomaterial passivation by an organized fibrin deposit and formation of EC monolayer on top achieve an optimal hemocompatibility without any modification of pericardial tissue.

Construction of a bilayered vascular graft with smooth internal surface for improved hemocompatibility and endothelial cell monolayer formation

Formation of a continuous endothelial cell (EC) monolayer inside the luminal surface with enhanced hemocompatibility, is a promising solution for improving performance and overall patency of small-diameter vascular grafts. There has been much debate regarding the ideal substrate surface topography to achieve this. Here, we investigated different polycaprolactone (PCL)-derived substrate surfaces fabricated from nanofibers, microfibers, or with smooth surfaces, and evaluated their effect on hemocompatibility and EC behavior. Our results demonstrated that, compared with the other two substrates, smooth surfaces inhibited platelet adhesion and activation, suppressed fibrinogen adsorption, and enhanced EC monolayer formation. In addition, smooth surfaces increased EC nitric oxide (NO) production and acetylated low-density lipoprotein (Ac-LDL) uptake. Thus, we designed and fabricated a bi-layered vascular graft composed of a smooth ultrathin internal layer and a microfibrous external layer, which were capable of preventing spiral-flow and decreasing wall-shear stress. Arterio-venous shunt models revealed that bi-layered grafts avoided bleeding and inhibited both protein absorption and platelet adhesion. Overall, these findings indicated that the prepared bi-layered grafts could perform better for in vivo implantation. Furthermore, this approach doesn't require chemical or biological modifications, and therefore can be easily applied to the fabrication of other implantable tubular grafts using various materials.

Mechanical Adaptability of the MMP-Responsive Film Improves the Functionality of Endothelial Cell Monolayer.

Extracellular matrix and cells are inherent in coordinating and adapting to each other during all physiological and pathological processes. Synthetic materials, however, show rarely reciprocal and spatiotemporal responses to cells, and lacking self-adapting properties as well. Here, a mechanical adaptability based on the matrix metalloproteinase (MMPs) sensitive polyelectrolyte film is reported. Poly-lysine (PLL) and methacrylated hyaluronic acid (HA-MA) nanolayers are employed to build the thin film through the layer-by-layer assembly, and it is further crosslinked using MMP sensitive peptides, which endows the films with changeable mechanical properties in response to MMPs. It is demonstrated that stiffness of the (PLL/HA-MA) films increases with the crosslinking, and then decreases in response to a treatment of enzyme. Consequently, the crosslinked (PLL/HA-MA) films reveal effective growth of endothelial cells (ECs), leading to fast formation of EC monolayer. Importantly, significantly improved endothelial function of the EC monolayer, which is characterized by integrity, biomolecules release, expression of function related gene, and antithrombotic properties, is achieved along with the decrosslinking of the film because of EC-secreted MMPs. These results suggest that mechanical adaptability of substrate in Young's modulus plays a significant role in endothelial progression, which shows great application potential in tissue engineering, regenerative medicine, and organ-on-a-chip.

Effects of different components of the extracellular matrix on endothelialization.

The endothelialization of cardiovascular prostheses is known to improve their haemocompatibility. As such body-foreign materials often do not endothelialize spontaneously. A lot of in vitro studies are ongoing how endothelialization of biomaterials can be improved. In this study the influence of different components of a tissue-typical extracellular matrix (ECM) like laminin, fibronectin or gelatin on the formation of an endothelial cell monolayer and on the shear resistance of adherent cells on these substrates was studied.The study revealed that the density of human venous endothelial cells (HUVEC) monolayers differed markedly between cells grown on a natural ECM and cells grown on singularized components of an ECM (p < 0.001). Only HUVEC grown on laminin showed similar densities and a stress fiber pattern comparable to HUVEC grown on the ECM. HUVEC grown on gelatin- or fibronectin-coated coverslips were less firmly attached to the substrate; frequently individual HUVEC and even groups of cells detached.Concluding it seems that coating of implants with laminin supports the formation of shear resistant endothelial cell (EC) monolayer - superior to other ECM components.

In Vitro Topographical Model of Fuchs Dystrophy for Evaluation of Corneal Endothelial Cell Monolayer Formation

A common indication for corneal transplantation, which is the most transplanted tissue, is a dysfunctional corneal endothelium due to Fuchs' endothelial dystrophy (FED). FED is diagnosed by the presence of in vivo pathological microtopography on the Descemet membrane, which is called corneal guttata. Minimally invasive corneal endothelial cell regenerative procedures such as endothelial cell injection therapy and Rho kinase inhibitor pharmacotherapy have been proposed as alternatives to conventional corneal transplantation for FED patients. However, the effect of guttata on monolayer reformation following such therapies is unknown and there is no equivalent in vitro or animal model to study monolayer reformation. Using a synthetic guttata FED disease model, the formation of the monolayer is investigated to evaluate the efficacy of both therapies. Results obtained suggest that guttata dimensions, density, and spacing greatly affect the fate of corneal endothelial cells in terms of migratory behavior and monolayer reformation. Densely packed synthetic guttata mimicking late-stage FED hinders monolayer reformation, while synthetic guttata of lower height and density show improved monolayer formation. These results suggest that severity of the FED, as determined by height and density of existing guttata, can potentially attenuate corneal endothelial monolayer formation of corneal cell injection therapy and pharmacotherapy.

Vascular Endothelial Growth Factor Improves Physico-Mechanical Properties and Enhances Endothelialization of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/Poly(ε-caprolactone) Small-Diameter Vascular Grafts In vivo

The combination of a natural polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and a synthetic hydrophobic polymer poly(ε-caprolactone) (PCL) is promising for the preparation of biodegradable and biocompatible small-diameter vascular grafts for bypass surgery. However, physico-mechanical properties and endothelialization rate of PHBV/PCL grafts are poor. We suggested that incorporation of vascular endothelial growth factor (VEGF) into PHBV/PCL grafts may improve their physico-mechanical properties and enhance endothelialization. Here we compared morphology, physico-mechanical properties, and in vivo performance of electrospun small-diameter vascular grafts prepared from PHBV/PCL with and without VEGF. Structure of the graft surface and physico-mechanical properties were examined by scanning electron microscopy and universal testing machine, respectively. Grafts were implanted into rat abdominal aorta for 1, 3, and 6 months with the further histological, immunohistochemical, and immunofluorescence examination. PHBV/PCL grafts with and without VEGF were highly porous and consisted mostly of nanoscale and microscale fibers, respectively. Mean pore diameter and mean pore area were significantly lower in PHBV/PCL/VEGF compared to PHBV/PCL grafts (1.47 μm and 10.05 μm2; 2.63 μm and 47.13 μm2, respectively). Durability, elasticity, and stiffness of PHBV/PCL grafts with VEGF were more similar to internal mammary artery compared to those without, particularly 6 months postimplantation. Both qualitative examination and quantitative image analysis showed that three-fourths of PHBV/PCL grafts with VEGF were patent and had many CD31-, CD34-, and vWF-positive cells at their inner surface. However, all PHBV/PCL grafts without VEGF were occluded and had no or a few CD31-positive cells at the inner surface. Therefore, VEGF enhanced endothelialization and improved graft patency at all the time points in a rat abdominal aorta replacement model. In conclusion, PHBV/PCL grafts with VEGF have better biocompatibility and physico-mechanical properties compared to those without. Incorporation of VEGF improves graft patency and accelerates formation of endothelial cell monolayer.

Improved Endothelial Function of Endothelial Cell Monolayer on the Soft Polyelectrolyte Multilayer Film with Matrix-Bound Vascular Endothelial Growth Factor.

Endothelialization on the vascular implants is of great importance for prevention of undesired postimplantation symptoms. However, endothelial dysfunction of regenerated endothelial cell (EC) monolayer has been frequently observed, leading to severe complications, such as neointimal hyperplasia, late thrombosis, and neoatherosclerosis. It has significantly impeded long-term success of the therapy. So far, very little attention has been paid on endothelial function of EC monolayer. Bioinspired by the microenvironment of the endothelium in a blood vessel, this study described a soft polyelectrolyte multilayer film (PEM) through layer-by-layer assembly of poly(l-lysine) (PLL) and hyaluronan (HA). The (PLL/HA) PEM was chemically cross-linked and further incorporated with vascular endothelial growth factor. It demonstrated that this approach could promote EC adhesion and proliferation, further inducing formation of EC monolayer. Further, improved endothelial function of the EC monolayer was achieved as shown with the tighter integrity, higher production of nitric oxide, and expression level of endothelial function related genes, compared to EC monolayers on traditional substrates with high stiffness (e.g., glass, tissue culture polystyrene, and stainless steel). Our findings highlighted the influence of substrate stiffness on endothelial function of EC monolayer, giving a new strategy in the surface design of vascular implants.

Evaluation of the corneal endothelial cell monolayer formation on synthetic guttata micro-structures to study the feasibility of corneal cell therapy

Event Abstract Back to Event Evaluation of the corneal endothelial cell monolayer formation on synthetic guttata micro-structures to study the feasibility of corneal cell therapy Muhammad Rizwan1, 2, Alon Ramos Mendez1, Khadijah Adnan3, Gary S. Peh3, 4, Wui Siew Tan2, Jodhbir S. Mehta4, 5 and Evelyn Yim1, 6, 7 1 National University of Singapore, Department of Biomedical Engineering, Singapore 2 Agency for Science, Technology and Research (A*STAR, Institute of Materials Research and Engineering, Singapore 3 Singapore Eye Research Institute, Tissue Engineering and Stem Cell Group, Singapore 4 Duke-NUS Graduate Medical School, Singapore 5 Singapore National Eye Centre, Singapore 6 National University of Singapore, Department of Surgery, Singapore 7 National University of Singapore, Mechanobiology Institute, Singapore Introduction: Majority of corneal transplants are performed exclusively to replace a dysfunctional corneal endothelium (CE) due to Fuchs’ endothelial dystrophy (FED)[1]. Due to shortage of donor corneas, corneal cell injection therapy has been proposed as a minimally invasive alternative treatment for FED patients[2]-[4]. A significant clinical sign of FED is the presence of excrescences of membrane (DM) which are called corneal guttata (CG)[5]-[7]. Current studies on cell injection therapy are performed in animal model corneas which have unaltered DM and hence, do not recapitulate the topographical micro-environment of FED[2][4],[8],[9]. Studying the monolayer formation on guttata-laden surface will be key to the development of cell therapy as a viable clinical procedure for corneal patients. The aim of this study was to fabricate the synthetic guttata-like micro-structures based on patient guttata characterization and evaluate the feasibility of cell monolayer formation on a synthetic guttata-laden surface for cell therapy applications. Material and Methods: Posterior corneal surfaces of FED patients were analyzed by using specular and transmission electron microscopy to characterize the CG dimensions. Soft lithography and hot embossing were used to fabricate similar dimensions guttata on polystyrene (synthetic guttata). The cell migration and monolayer formation on synthetic guttata was studied by first using B4G12 corneal cell line on various dimensions of guttata followed by primary human corneal endothelial cells (HCECs) on selected guttata. Cell interaction with synthetic guttata was studied with SEM imaging and monolayer formation was studied by using confocal microscopy in terms of ZO1 expression. Cell migration tracks were plotted by using manual tracking plugin in ImageJ and analyzed with Ibidi chemotaxis tool. Results and Discussion: B4G12 cell monolayer formation on top of synthetic guttata pillars gradually increased as the pillar spacing and diameter was increased (Fig. 1). The cell morphology was elongated instead of polygonal on highest density pillars. B4G12 cell migration distance, speed and directness analysis on synthetic guttata pillars showed that the highest density pillars presented the most challenging surface for cell monolayer formation, ZO1 expression and for cell migration. With primary HCECs, the cells were unable to form monolayer on highest density small pillars with migrated cells or with cell seeded on top of pillars. Decreasing the height and the diameter and making the edges round (lenses) led to an increased ZO1 expression on top of the micro-structures which indicated that the cells could form tight junction on synthetic guttata (Fig. 2). HCECs migration analysis showed that the synthetic guttata with round edges and less height (5µm) were relatively more favorable for cell migration and corneal endothelial monolayer formation (Fig. 3). The results suggest that the diseased guttata-laden corneal DM surface might potentially hinder the formation of corneal endothelium. Conclusion: The results of this study shows that the pre-existing DM guttata could interfere with injected cells and corneal endothelial monolayer formation within eye. The surgical removal of preexisting guttata before cell therapy could potentially increase the chance of cell therapy success. Additionally, the characterization of the types of guttata on corneal DM surface of FED patients could also be important for patient-specific cell therapy procedure. MOE AcRF Tier 1 – FRC R-397-000-217-112; National Research Foundation Translational and Clinical Research (TCR) Programme Grant (NMRC/TCR/008-SERI/2013); National Medical Research Council NMRC/NIG/0037/2008; Mechanobiology Institute (MBI) R-714-007-006-271; Biomedical Research Council Translation Clinical Research Partnership (TCRP) Grant (TCR0101673); Singapore International Graduate Award (SINGA) Scholarship by A*STAR Graduate Academy

Micro- and nano-topography to enhance proliferation and sustain functional markers of donor-derived primary human corneal endothelial cells

One of the most common indications for corneal transplantation is corneal endothelium dysfunction, which can lead to corneal blindness. Due to a worldwide donor cornea shortage, alternative treatments are needed, but the development of new treatment strategies relies on the successful in vitro culture of primary human corneal endothelial cells (HCECs) because transformed cell lines and animal-derived corneal endothelial cells are not desirable for therapeutic applications. Primary HCECs are non-proliferative in vivo and challenging to expand in vitro while maintaining their characteristic cell morphology and critical markers. Biochemical cues such as growth factors and small molecules have been investigated to enhance the expansion of HCECs with a limited increase in proliferation. In this study, patterned tissue culture polystyrene (TCPS) was shown to significantly enhance the expansion of HCECs. The proliferation of HCECs increased up to 2.9-fold, and the expression amount and localization of cell–cell tight junction protein Zona Occludens-1 (ZO-1) was significantly enhanced when grown on 1μm TCPS pillars. 250nm pillars induced an optimal hexagonal morphology of HCEC cells. Furthermore, we demonstrated that the topographical effect on tight-junction expression and cell morphology could be maintained throughout each passage, and was effectively ‘remembered’ by the cells. Higher amount of tight-junction protein expression was maintained at cell junctions when topographic cues were removed in the successive seeding. This topographic memory suggested topography-exposed/induced cells would maintain the enhanced functional markers, which would be useful in cell-therapy based approaches to enable the in situ endothelial cell monolayer formation upon delivery. The development of patterned TCPS culture platforms could significantly benefit those researching human corneal endothelial cell cultivation for cell therapy, and tissue engineering applications.

Oxygen‐rich coating promotes binding of proteins and endothelialization of polyethylene terephthalate polymers

The formation of endothelial cell monolayer on prosthetic implants has not sufficiently explored. The main reasons leading to the development of thrombosis and/or intimal hyperplasia is the lack of endothelialization. In the present work, we have studied the influence of oxygen and fluorine plasma treatment of polyethylene terephthalate (PET) polymers on human microvascular endothelial cell adhesion and proliferation. We characterized the polymer surface, wettability, and oxidation potential upon plasma treatment. Moreover, binding of serum and media compounds on PET surface was monitored by Quartz crystal microbalance method, X-ray photoelectron spectroscopy, and atomic force microscopy. Cell adhesion and morphology was assessed by light and scanning electron microscopy. The influence of plasma treatment on induction of cellular oxidative stress and cell proliferation was evaluated. The results obtained showed that treatment with oxygen plasma decreased the oxidation potential of the PET surface and revealed the highest affinity for binding of serum components. Accordingly, the cells reflected the best adhesion and morphological properties on oxygen-treated PET polymers. Moreover, treatment with oxygen plasma did not induce intracellular reactive oxygen species production while it stimulated endothelial cell proliferation by 25% suggesting the possible use of oxygen plasma treatment to enhance endothelialization of synthetic vascular grafts.

Effects of irradiated biodegradable polymer in endothelial cell monolayer formation

In this work we study cell adhesion, proliferation and cell morphology of endothelial cell cultured on poly-l-lactide acid (PLLA) modified by heavy ion irradiation. Thin films of PLLA samples were irradiated with sulfur (S) at energies of 75MeV and gold (Au) at 18MeV ion-beams. Ion beams were provided by the Tandar (Buenos Aires, Argentina) and Tandetron (Porto Alegre, Brazil) accelerators, respectively. The growth of a monolayer of bovine aortic endothelial cells (BAEC) onto unirradiated and irradiated surfaces has been studied by in vitro techniques in static culture. Cell viability and proliferation increased on modified substrates. But the results on unirradiated samples, indicate cell death (necrosis/apoptosis) with the consequent decrease in proliferation. We analyzed the correlation between irradiation parameters and cell metabolism and morphology.

P 13: Chemokine receptor antagonists influence on monocytes infiltration through endothelial cells monolayer studied by a real-time electrical impedance assay

Background and aims Atherosclerosis is characterized by an inflammatory process and a specific repertoire of chemokines and cell adhesion molecules expressed by endothelial cells (EC) that attract leukocytes to the developing atheroma. The inhibition of chemokine/chemokine receptor interactions and prevention of accumulation of pro-inflammatory cells in the vascular wall may constitute an important therapeutic option. Our aim was to follow in real-time the effect of chemokine receptor antagonists (CRA) on monocyte adhesion and transmigration through endothelial cell monolayer. Materials and Methods We used a method based on changes in electrical impedance to study in real-time the monocytes infiltration through endothelial cell monolayer. The endothelial cells (line EA.hy926) were cultured in 16-well e-plates covered with gold microelectrode that monitors and quantifies the infiltration of human monocytes (U937 cell line) in the presence/absence of CRA specific to chemokine receptors CCR2 (Teijin1 coumpound 1 and BMS CCR2) and CCR5 (maraviroc) and a broad-range CRA (pertussis toxin). Results The formation of EC monolayer was documented by the increase in electrical impedance in the first 24 hours of the experiments. The infiltration of monocytes, characterized by the opening of endothelial cell junctions, retraction of endothelial monolayer and replacement by monocytes determined a significant decrease in impedance. In contrast, the incubation of EC with monocytes in the presence of chemokine receptors antagonists lead to increases in impedance values indicating that monocytes infiltration was inhibited in the presence of CRA. The percentage of increases in impedance were calculated at different time points over three days and at the end of experiment the values were as follows: for Teijin compound 1 by ∼ 17%, for BMS CCR2 an maraviroc by ∼30% and for PTX by ∼ 20% Conclusion This novel technique monitors in real-time the effect of CRA on adhesion and transmigration of monocytes through endothelial monolayer. The advantages of this method in comparison with other methods (Boyden chamber, matrigel assay) are: 1) the endothelial cell-monocyte interaction mimics more closely the in vivo process, and 2) the data are obtained in real-time and are more reliable quantify as opposed to end-point analysis for other methods.

Engineering high-density endothelial cell monolayers on soft substrates

This study demonstrates that a confluent monolayer of endothelial cells (ECs) can be tissue engineered on a soft substrate with a cell density and morphology that approximates in vivo conditions. We achieved formation of a confluent EC monolayer on polydimethylsiloxane (PDMS) elastomer by microcontact printing of fibronectin (FN) in a square lattice array of 3 μm diameter circular islands at a 6 μm pitch. Uniform coatings of FN or serum proteins on PDMS or on tissue-culture-treated polystyrene failed to support the equivalent EC density and/or confluence. The ECs on the FN micropatterned PDMS achieved a density of 1,536 ± 247 cells mm −2, close to the 3,215 ± 336 cells mm −2 observed in vivo from porcine pulmonary artery and significantly higher (2- to 5-fold) than EC density on other materials. The probable mechanism for enhanced EC adhesion, growth and density is increased focal adhesion (FA) formation between the ECs and the substrate. After 14 days culture, the micropatterned FN surface increased the average number of FAs per cell to 35 ± 10, compared to 7 ± 6 for ECs on PDMS uniformly coated with FN. Thus, microscale patterning of FN into FA-sized, circular islands on PDMS elastomer promotes the formation of EC monolayers with in vivo-like cell density and morphology.

Cultivation of Endothelial Progenitor Cells on Fibrin Matrix and Layering on Dacron/Polytetrafluoroethylene Vascular Grafts

Completely biological tissue-engineered vascular graft is an upcoming substitute for damaged blood vessel, but its clinical use is currently limited due to poor mechanical strength. Therefore, at present, polymeric small-diameter vascular grafts lined with endothelial cells (ECs) to reduce graft thrombosis may be a more viable option. Successful construction of EC-seeded artificial grafts faces some challenges such as (i) retention of endothelial lining; and (ii) availability of differentiated autologous cells. Biomaterial surfaces that are modified by depositing extracellular matrix (ECM) components may stabilize cells in the lumen against forces of blood flow. Adult stem cells such as endothelial progenitor cells (EPCs) circulate in the blood and they usually attach to the exposed matrix at the injured blood vessel site. Depending on the signaling capabilities of ECM, cells may differentiate into ECs,, and if a similar composition of the matrix is provided in vitro, EPCs isolated from blood might get differentiated and thus autologous cells for tissue engineering may be obtained. In this in vitro study, ECM scaffold consisting of biomolecules such as fibrin, fibronectin, and gelatin along with growth factors is found to have supported differentiation of EPC into EC. Further, the ECM precoated on Dacron and polytetrafluoroethylene is found to have supported the formation of EC monolayer that synthesized nitric oxide, and resisted shear stress. Thus, biomimetic fibrin composite is found to be suitable not only to seed cells on currently available artificial grafts but also to obtain differentiated EC from EPC.

Isolation and characterization of PECAM‐1‐deficient retinal endothelial cells expressing a specific isoform of PECAM‐1

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is expressed at high levels on endothelial cells. Alternative splicing of PECAM-1 generates multiple isoforms, which differ in the length of their cytoplasmic domains. We have shown that multiple isoforms of PECAM-1 are expressed in vascular beds of different tissues and endothelial cells; and their expression is regulated during vascular development and angiogenesis. However, how these isoforms differ in their adhesive and signaling properties remains largely unknown. Previous studies using PECAM-1 antibodies have demonstrated a role for PECAM-1 in endothelial cell monolayer formation, capillary morphogenesis in Matrigel, and angiogenesis. The PECAM-1 deficient (PECAM-1 -/-) mice have been generated and show a number of vascular abnormalities during angiogenesis and inflammatory challenges. The role of PECAM-1 isoforms in these processes requires further investigation. Here we describe the isolation of conditionally immortalized endothelial cells from the retinas of wild type and PECAM-1 -/- mice. We show that PECAM-1 -/- endothelial cells are less migratory and fail to undergo capillary morphogenesis in Matrigel. Expression of PECAM-1 in these cells restores these defects in an isoform specific manner. We show that expression of the PECAM-1 isoform lacking exons 14 and 15, but not the isoform that lacks exon 15, enhances the migration and capillary morphogenesis of PECAM-1 -/- retinal endothelial cells. These results demonstrate PECAM-1 functions that are essential during vascular development and angiogenesis; and are regulated by alternative splicing of its cytoplasmic domain. This work was supported by the American Heart Association Predoctoral Fellowship 0510047Z.

Endothelial cell monolayer formation: effect of substrate and fluid shear stress.

The formation of an endothelial cell (EC) monolayer is one critical factor in the development of a tissue engineered vascular graft. One potential method of endothelialization is the migration of native ECs from the surrounding blood vessel onto the newly implanted graft. In the present study, in vitro experiments were performed to investigate the potential of ECs to migrate on a tissue-engineered blood vessel wall model (TEWM) and form a new monolayer. The TEWM was composed of a three-dimensional, type I collagen matrix seeded with smooth muscle cells. The potential of ECs to form new monolayer was evaluated in the absence and presence of fluid shear stress (10 dynes/cm(2)). The monolayer formation on the TEWM was compared to a control, two-dimensional substrate of glass coated with type I collagen. Results from these studies showed that EC monolayer formation was inhibited on the TEWM in comparison to glass coated with collagen. This correlated with an inhibition of proliferation on the TEWM. The application of shear stress to the EC on the glass coated with collagen also caused an inhibition of monolayer formation, with a corresponding inhibition of proliferation. Furthermore, blocking proliferation by incubation with mitomycin C caused a dose-response inhibition of monolayer formation. In contrast, stimulating proliferation with basic fibro- blast growth factor (bFGF) did not further increase monolayer formation on glass coated with collagen. These results suggest that proliferation is one necessary factor for monolayer formation, although not the only factor, where EC proliferation is regulated by its environment, including both substrate and the local shear stress. Continued investigation into the mechanism and stimulation of EC proliferation on the TEWM may lead to developing new strategies for the endothelialization of a tissue engineered vascular graft.

The dense peripheral band: a microfilament structure associated with endothelial cell monolayer formation

The dense peripheral band: a microfilament structure associated with endothelial cell monolayer formation