Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25-100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.

Autocrine amplification of integrin αIIbß3 activation and platelet adhesive responses by deoxyribose-1-phosphate.

Using direct injection mass spectrometry (DIMS) we discovered that deoxyribose-1-phosphate (dRP) is released by platelets upon activation. Interestingly, the addition of exogenous dRP to human platelets significantly increased platelet aggregation and integrin αIIbß3 activation in response to thrombin. In parallel, genetically modified platelets with double genetic deletion of thymidine phosphorylase and uridine phosphorylase were characterised by reduced release of dRP, impaired aggregation and decreased integrin αIIbß3 activation in response to thrombin. In vitro platelet adhesion onto fibrinogen and collagen under physiological flow conditions was potentiated by treatment of human platelets with exogenous dRP and impaired in transgenic platelets with reduced dRP release. Human and mouse platelets responded to dRP treatment with a sizeable increase in reactive oxygen species (ROS) generation and the pre-treament with the antioxidant apocynin abolished the effect of dRP on aggregation and integrin activation. Experiments directly assessing the activation of the small G protein Rap1b and protein kinase C suggested that dRP increases the basal levels of activity of these two pivotal platelet-activating pathways in a redox-dependent manner. Taken together, we present evidence that dRP is a novel autocrine amplifier of platelet activity, which acts on platelet redox levels and modulates integrin αIIbß3.

A novel method for the extraction and culture of progenitor stem cells from human peripheral blood for use in regenerative medicine.

Human peripheral blood (HPB) contains both circulating endothelial cells (CECs) and endothelial progenitor stem cells (EPCs), which may be suitable for use in regenerative medicine. There has been considerable interest in using these cells, but there is no "gold standard" technique for isolating these cells. The aim of this study was to characterize and compare a number of different extraction and culture techniques to develop a system to isolate and culture cells. EPC and CEC were isolated from HPB using either Histopaque-1077 or Lymphoprep. The two isolation methods were compared for the number of cells isolated, cell metabolism, and RNA expression. Both isolations produced viable cells and were comparable. The tissue culture method employed does have a significant effect on the cell population with regard to medium choice, fetal bovine serum concentration, and surface modification of the culture surface. In conclusion, it can be seen that although this study and previous work can suggest a basis for culture, further work to develop an optimized and agreed "gold standard" culture regime for EPC from HPB is required to maximize the potential of this source of cells for regenerative medicine and to translate its clinical use in the future.

The long-term stability in gene expression of human endothelial cells permits the production of large numbers of cells suitable for use in regenerative medicine.

Tissue engineering has been conducted in the study of cardiovascular grafts for many years. Many obstacles have been overcome in this rapidly changing field, but one difficulty has remained until now: the large number of endothelial cells (ECs) needed for seeding the inner layer of bypass graft. Recent advances in endothelial progenitor cell (EPC) isolation and culture techniques have increased the interest in genetic studies. Despite these advances in EPC studies, the "gold standard" for the seeding of tissue engineering constructs or hybrid grafts remains mature human umbilical vein endothelial cells (HUVECs). This study investigates the ability of HUVECs to be expanded in culture to provide sufficient cells for graft seeding. The levels of gene expression of key genes are then examined to ensure that these cells retain the EC phenotype. This study demonstrates that HUVECs may be cultured for up to 12 passages without alteration in phenotype. Subsequent passage numbers are sufficiently similar to those preceding them to allow cells of different passages to be mixed without gene expression anomalies.

Haemodynamic regulation of gene expression in vascular tissue engineering.

Synthetic grafts, namely expanded polytetrafluoroethlene (ePTFE) and poly(ethylene terephthalate) (Dacron), used for cardiovascular bypass surgery are thrombogenic. Lining the inner lumen ("seeding") of synthetic grafts with endothelial cells (ECs) increases patency rates similar to those of autologous grafts (e.g. saphenous vein). The major drawback with seeding grafts is the retention of cells present on the graft after implantation in vivo, where large portions of cell wash off. Preconditioning the seeded EC monolayer with shear stress has been shown to promote the reorganisation of the EC cytoskeleton and production of extracellular matrix, resulting in higher EC retention after exposure to blood flow. Vascular ECs have a number of essential and complex roles. ECs synthesise and secrete vasoconstrictors, vasodilators, growth factors, fibrinolytic factors, cytokines, adhesion molecules, matrix proteins and mitogens that modulate many physiological processes such as wound healing, hemostasis, vascular remodelling, inflammatory and immune responses. Vascular cells in vivo are exposed to hemodynamic forces created by the pulsatile flow of blood through the vessel. Due to their unique anatomical position, ECs are constantly exposed to shear stress forces and allow the vessel wall to adapt to changes by modulating EC structure and function. This review describes the mainly in vitro and in vivo studies used to define the molecular role hemodynamics have in vascular disease and its usage in developing tissue engineered vascular bypass grafts.

In vitro small intestinal epithelial cell growth on a nanocomposite polycaprolactone scaffold.

Tissue engineering of the small intestine remains experimental despite worldwide attempts to develop a functional substitute for short bowel syndrome. Most published studies have reported predominant use of PLLA (poly-L-lactide acid)/PGA (polyglycolic acid) copolymer as the scaffold material, and studies have been limited by in vivo experiments. This lack of progress has inspired a fresh perspective and provoked further investigation and development in this field of tissue engineering. In the present paper, we exploit a relatively new nanocomposite of POSS (polyhedral oligomeric silsesquioxane) and PCL [poly(caprolactone-urea)urethane] as a material to develop porous scaffolds using a solvent casting/particulate leaching technique to fabricate porous scaffolds in different pore sizes and porosities. Scaffolds were characterized for pore morphology and porosity using scanning electron microscopy and micro-computed tomography. Rat intestinal epithelial cells were then seeded on to the polymer scaffolds for an in vitro study of cell compatibility and proliferation, which was assessed by Alamar Blue and lactate dehydrogenase assays performed for 21 days post-seeding. The results obtained demonstrate that POSS-PCL nanocomposite was produced as a macroporous scaffold with porosity over the range of 40-80% and pore size over the range of 150-250 microm. This scaffold was shown to support epithelial cell proliferation and growth. In conclusion, as a further step in investigating small intestinal tissue engineering, the nanocomposite employed in this study may prove to be a useful alternative to poly(lactic-co-glycolic acid) in the future.

Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures.

Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-D-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin alphavbeta3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.

Endothelial cell retention on a viscoelastic nanocomposite vascular conduit is improved by exposure to shear stress preconditioning prior to physiological flow.

In this study, endothelial cell (EC)-seeded nanocomposite grafts were preconditioned with 1-2 dynes/cm(2) in vitro to establish whether low shear stress resulted in improved cell adherence prior to physiological shear stress (15 dynes/cm(2)). Alamar blue cell viability was assessed. Polymerase chain reaction was conducted for glyceraldehyde-3-phosphate dehydrogenase, transforming growth factor beta-1 (TGFbeta-1), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet EC adhesion molecule-1, and vascular endothelial growth factor receptor-2 (VEGFR-2). The Alamar blue results demonstrated improved cellular retention following preconditioning (P < 0.001). VEGFR-2 and TGFbeta-1 expression was up-regulated, and VEGFR-1 down-regulated following preconditioning. This investigation confirms previous findings regarding the potential benefits of preconditioning, and demonstrates that these benefits can be applied to ECs seeded on the nanocomposite employed. It also demonstrates further the suitability and potential of nanocomposite for future use in tissue-engineered cardiovascular devices.

The effect of shear stress on human endothelial cells seeded on cylindrical viscoelastic conduits: an investigation of gene expression.

The present study assesses the effect of physiological shear stress on gene expression from human ECs (endothelial cells) seeded on a small-diameter cylindrical bypass graft constructed from nanocomposite based on poly(carbonate-silsesquioxane-bridge-urea)urethane. ECs were seeded on to 5-mm-diameter conduits, placed in a physiological flow circuit and exposed to 1 or 4 h of shear stress at 1.4+/-0.3 Pa. Subsets of conduits were incubated at 37 degrees C and 5% CO2/95% O2 for a further 4 h to determine if gene expression returned to basal levels. PCR was conducted for glyceraldehyde-3-phosphate dehydrogenase, TGFbeta-1 (transforming growth factor beta-1), COL-1 (collagen-1) and PECAM-1 (platelet/EC adhesion molecule-1). Increases in gene expression were seen following flow in nanocomposite conduits. These were significant at 4 h for TGFbeta-1, COL-1 and PECAM-1. After a 4 h recovery period, there were no significant differences in gene intensity, suggesting that this change is transient. These data prove that mRNA can be obtained from ECs seeded on tubular conduits and exposed to shear stress and that gene-expression studies can be successfully carried out. We believe this is a substantial improvement on studies based on flat sheets.

Review paper: principles and applications of surface analytical techniques at the vascular interface.

Surface properties have been found to be one of the key parameters which cause degradation and of thrombogenicity in all polymers used in biomedical devices, thus signifying the importance and the necessity for quantitative and accurate characterization of the polymer surface itself as used in the construction of the device. The characterization techniques employed generally involve thermal and spectroscopic measurements, in which class the electrochemical investigations and scanning probe microscopies can also be included. Current hypotheses on the correlations that exist between surface parameters and hemocompatibility and degradation of polymers are examined herein, but concentrating on the field of clinically utilized polymeric materials as used within medical devices themselves. Furthermore, this review provides a brief but complete synopsis of these techniques and other emerging ones, which have proven useful in the analysis of the surface properties of polymeric materials as used in the construction of cardiovascular devices. Statements and examples are given as to how specific information can be acquired from these differing methodologies and how it aids in the design and development of new polymers for usage in biomedical device construction.

Cardiovascular tissue engineering: state of the art.

In patients requiring coronary or peripheral vascular bypass procedures, autogenous arterial or vein grafts remain as the conduit of choice even in the case of redo patients. It is in this class of redo patients that often natural tissue of suitable quality becomes unavailable; so that prosthetic material is then used. Prosthetic grafts are liable to fail due to graft occlusion caused by surface thrombogenicity and lack of elasticity. To prevent this, seeding of the graft lumen with endothelial cells has been undertaken and recent clinical studies have evidenced patency rates approaching reasonable vein grafts. Recent advances have also looked at developing a completely artificial biological graft engineered from the patient's cells with surface and viscoelastic properties similar to autogenous vessels. This review encompasses both endothelialisation of grafts and the construction of biological cardiovascular conduits.

Interactions between endothelial cells and a poly(carbonate-silsesquioxane-bridge-urea)urethane.

We have recently developed a polymer which contains silsesquioxane in the form of nano-bridges poly(carbonate-silsesquioxane-bridge-urea)urethane (PCBSU) for cardiovascular device applications. The polymer has been characterised and the durability has been confirmed with long-term in vivo tests. The aim of this study was to test the cytocompatibility of the new polymer and to investigate any potential cytotoxic effects. To assess the effect of direct contact with PCBSU sections of polymer material were cut and placed into a 24-well plate. Six discs were seeded with 2 x 10(5) human umbilical vein cells (HUVEC). As a positive control, six wells were seeded with the same number of HUVEC. In a further experiment to assess indirect contact with PCBSU a sample of the polymer was powdered using a Micro-Dismembrator. Cell culture medium was exposed to powdered polymer (1-100 mg/ml) for a period of 7 days. HUVEC seeded as above were then exposed to the treated cell culture medium for 24 and 96 h. Finally, cell proliferation was studied over 16 days by seeding 2 x 10(5) HUVEC on films of PCBSU cast in glass Petri dishes. Cell viability and growth were assessed using Alamar blue, lactate dehydrogenase and Pico green assays and morphology was studied by Toluidine blue staining and scanning electron microscopy. Viable cells were demonstrated to be present after 16 days seeded on PCBSU. Exposing cells to PCBSU-treated cell culture medium resulted in no apparent damage to the cells at concentrations of 1 or 10 mg/ml, and only a slight reduction at 100 mg/ml after 96 h exposure. This study demonstrates that PCBSU can support the growth of endothelial cells for a prolonged period and does not demonstrate any significant toxic effects to cells. Thus it has the potential to be used both as a medical device and as scaffolding in tissue engineering applications.

Development of an RNA isolation procedure for the characterisation of human endothelial cell interactions with polyurethane cardiovascular bypass grafts.

To date no reliable method has been developed for the isolation of RNA from cells seeded onto cylindrical vascular grafts. This study was performed in order to develop a reliable methodology for isolating RNA from cylindrical conduits made from poly(carbonate-urea)urethane (PU). Human umbilical vein EC were seeded onto PU vascular grafts and an Alamar blue assay performed to assess cell viability. Cells were prepared for RNA extraction by trypsinisation, cell scraping and direct application of cell lysis buffer. In all cases RNA was extracted using a "Qiagen RNeasy" kit. Alamar blue showed viable cells were present on all of the seeded PU vascular grafts. Levels of RNA extracted from the cells removed from the graft by the trypsinisation yielded 0.130 microg/microl, by scraping 0.078 microg/microl and by direct lysing 0.093 microg/microl of RNA, respectively. RTPCR was conducted successfully for GAPDH and TGF-beta1. Trypsinisation prior to RNA extraction provided the highest RNA yield and attained near complete cell removal ensuring that gene expression obtained was representative.