European Journal of Vascular & Endovascular Surgery
Volume 32, Issue 1 , Pages 76-83, July 2006

Nanocomposite Containing Bioactive Peptides Promote Endothelialisation by Circulating Progenitor Cells: An In vitro Evaluation☆

  • N. Alobaid

      Affiliations

    • Academic Division of Surgical and Interventional Sciences, Biomaterials and Tissue Engineering Centre (BTEC), University College London, Hampstead Campus, London NW3 2PF, UK
    • ,
  • H.J. Salacinski

      Affiliations

    • Academic Division of Surgical and Interventional Sciences, Biomaterials and Tissue Engineering Centre (BTEC), University College London, Hampstead Campus, London NW3 2PF, UK
    • ,
  • K.M. Sales

      Affiliations

    • Academic Division of Surgical and Interventional Sciences, Biomaterials and Tissue Engineering Centre (BTEC), University College London, Hampstead Campus, London NW3 2PF, UK
    • ,
  • B. Ramesh

      Affiliations

    • Department of Protein and Molecular Biology, University College London, Hampstead Campus, London NW3 2PF, UK
    • ,
  • R.Y. Kannan

      Affiliations

    • Academic Division of Surgical and Interventional Sciences, Biomaterials and Tissue Engineering Centre (BTEC), University College London, Hampstead Campus, London NW3 2PF, UK
    • ,
  • G. Hamilton

      Affiliations

    • Vascular Unit, Royal Free Hampstead NHS Trust Hospital, London NW3 2QG, UK
    • ,
  • A.M. Seifalian

      Affiliations

    • Academic Division of Surgical and Interventional Sciences, Biomaterials and Tissue Engineering Centre (BTEC), University College London, Hampstead Campus, London NW3 2PF, UK
    • Corresponding Author InformationCorresponding author. Professor Alexander M. Seifalian, PhD, Academic Division of Surgical and Interventional Sciences, Biomaterials and Tissue Engineering Centre, University College London, Rowland Hill Street, Hampstead, London NW3 2PF, UK.

Accepted 13 November 2005. published online 08 February 2006.

Article Outline

Abstract 

Objective

The formation of an endothelial cell layer on the luminal surface of cardiovascular devices, especially bypass grafts, is an important attribute in order to improve their patency. Endothelial progenitor cells (EPCs) have a potential role in the endothelialisation of bypass grafts. We hypothesised that a novel approach to improve endothelialisation of bypass grafts by EPCs would be the creation on the graft lumen of a microenvironment that supports EPC adhesion and differentiation.

Methods

A new generation of nanocomposite based on silsesquioxane in the form of polyhedral oligomeric silsesquioxane (POSS) nanocages which incorporate bioactive peptides (RGD) was made into sheets. Peripheral blood mononuclear cells (PBMCs) containing EPCs isolated from six consenting young, healthy, adult volunteers were then plated both on (1) sheets of the nanocomposite with the bioactive peptide, (2) sheets of the nanocomposite without the bioactive peptide, (3) culture dishes as control and then cultured in presence of vascular endothelial growth factor (VEGF). Confirmation of endothelial and EPCs markers was carried out using fluorescence-activated cell sorter (FACS) analysis, reverse transcription polymerase chain reaction (RT-PCR) and immunostaining.

Results

One to two percent of PBMCs expressed CD34 as determined by FACS analysis. Cells were demonstrated to express mRNA for the EPC markers CD34, platelet-endothelial cell adhesion molecule-1 (CD31), CD133 and vascular endothelial growth factor receptor-2(FlK-1/KDR). Endothelial cell-colony forming units were formed between day 5 and day 7 after plating. Colonies were confirmed to be endothelial like cells by immunostaining. There were significantly greater numbers of EPC colonies on the bioactive nanocomposites as compared to the nanocomposite alone and the uncoated dishes.

Conclusion

We report a new nanocomposite based biomaterial that has been demonstrated, in vitro, to promote endothelialisation from PBMCs containing EPCs.

Keywords: Nanocomposite, Tissue engineering, Stem cells, Endothelial progenitor cells, Bypass graft, Peptides, Vascular, RGD, silsesquioxane

 

Back to Article Outline

1. Introduction 

Bypass grafts commonly are utilised for the treatment of coronary or peripheral atherosclerosis, principally using autogenous tissue. In 30–50% bypass patients, all viable autologous tissue has already been used;1 under these circumstances synthetic prostheses are employed. The current clinical prosthetic conduits are based on either expanded polytetrafluroethylene (ePTFE) or polyethylene terephthalate (Dacron). However, patency at 5 years is only 40–50% when used to bypass to the proximal popliteal artery and 20% at 3 years when used for infra-popliteal bypass.2

To improve the patency rate such prostheses have been seeded with endothelial cells using a two-stage procedure.3 Two-stage seeding requires the harvesting of endothelial cells from a vein biopsy, followed by culturing/amplification for 2–4 weeks and then seeding onto an ePTFE graft coated with the synthetic peptide arginine-glycine-aspartic acid (RGD) and fibrin. Clinical trials using endothelialised PTFE grafts for femoro-popliteal bypass have been promising. In a phase 1 randomised study comparing endothelialised PTFE grafts to controls, Zilla and colleagues reported 5-year patency rates of 73.8% as compared to 31.3% in control(s).4 In a more recent paper,5 they reported the results of 153 such endothelialised grafts with a primary patency of 62.8% at 7 years. Interestingly, this represented a patency rate of 70.8% for bypass to vessels below the knee and 60% for those above knee. However, such surgery is limited to a few specialised centres with the necessary cell culture facilities, technicians and bioreactors.

The discovery that vascular endothelial ‘stem cells’ or progenitor cells (EPC) exist,6 even in patients suffering from cardiovascular disease, has led to interest in their use in tissue engineering. The relative ease of isolating these cells and their capacity to be expanded in culture, up to as many as 1000 doublings, whilst retaining their capacity to differentiate7 these studies make EPC an interesting cell type for use in tissue engineering. Increasing evidence indicates that EPC have the potential to differentiate into mature endothelial cells and contribute to the process of endothelium repair, as they can home to specific sites in response to endothelial injury, where they can divide.8

The number of circulating EPC increase in response to local ischaemia,9 statins,10 oestrogen,11 erythropoietin12 and vascular endothelial growth factor (VEGF). There is a rapid mobilisation of EPCs after burn injury and surgical manipulation.13 Raised EPCs can be detected in the circulation 6h after injury and by 24h EPCs constitute 12% of total circulating mononuclear blood cells.14

EPCs have been differentiated into endothelial cells in vitro and incorporated into sites of neovascularisation in vivo. EPCs show less plasticity and demonstrate less capacity for self-renewal than earlier stem cells, but retain the ability to differentiate into several cell types. EPC differentiation in vitro is dependent on culture conditions. VEGF and fibronectin are reported to promote the differentiation of EPC into endothelial cells.15 EPC are a heterogeneous groups of cells and the exact definitions of all cells with endothelial progenitor potential are still elusive.16 Several different types of cells have been defined as EPC. (1) CD14 expressing monocytes/macrophages and mostly negative for stem cell markers like CD34 and CD133.17, 18 (2) Cells possessing markers like CD34 and CD 133 but do not express CD14 and probably lose CD133 and CD34 as they mature. So for the purpose of this paper we used the term EPC to describe any cell that has the potential to develop into a cell expressing endothelial cell markers. At this point we do not exclude the presence of mature endothelial cells within the circulation. These may have sloughed off of the vessel walls. We would not expect such cells to be useful but we cannot exclude the presence of these cells based on this study.

Nanocomposites are materials whose components exist at a nanometer scale. This nature allows the resulting material to exhibit different properties from its conventional bulk counterpart (micro-composites).19 In microcomposites phase separation between constituents is more prominent while in nanocomposites, there is increased intercalation between the layers of the matrix or even further dispersion uniformly within the matrix to form an exfoliated type nanostructure.

Once formed nanocomposites exist as stacked layers separated by an interface termed ‘interlayer’ or ‘gallery’. Depending on the ultra-structure of the nanocomposite they are classified as follows.20 Type I: organic polymer embedded in an inorganic matrix without covalent bonding between the components; type II: organic polymer embedded in an inorganic matrix with sites of covalent bonding between the components; type III: co-formed interpenetrating networks of inorganic and organic polymers without covalent bonds between phases; type IV: co-formed interpenetrating networks of inorganic and organic polymers with covalent bonds between phases and type V: non-shrinking simultaneous polymerization of inorganic and organic polymers.

Due to the size of these nano-fillers, their surface areas are up to 400% more than conventional microcomposites and as such they dramatically increase the polymer mechanical strength. We have shown that by direct synthesis of these nanofillers into a polymer, in this case carbonate-based polyurethanes produce strong, bio-durable polymers.20, 21 Our original hypothesis was that by incorporation of nanocomposites into a carbonate based polyurethane a novel class of biomaterial would be generated, which exhibits unique biological properties previously unattainable due principally to their non-linearity.20 These properties engender amphiphilic and thermodynamic qualities. In this study we utilised a novel type of silica-based nanocomposite termed polyhedral oligomeric silsesquioxanes (POSS). We have shown that polyurethanes containing POSS result in materials with anti-platelet, coagulation inhibitory (heparin-like effect) and anti-infection (bacterial adhesion and growth prevention) properties.22

Extensive research over the last decade has been performed on the incorporation of adhesion promoting peptides into biomaterial surfaces. Since, identification of the RGD peptide sequence as mediating the attachment of cells to several plasma and ECM proteins, including fibronectin and vitronectin, researchers have been incorporating Arg-Gly-Asp (RGD)-containing peptides on biomaterials to promote cell attachment.23

The peptide GRGD in solution inhibits fibrinogen binding to endothelial cells and fibrinogen-induced endothelial cell(s) migration.23 When this peptide was photochemically grafted to the surface of a polyethylene glycol modified polyurethane (PU–PEG) to form PU–PEG–GRGD, endothelial cell adhesion and proliferation was improved. The enhancement efficiency was correlated with GRGD content.3 In similar studies coupling an ePTFE graft surface with RGD-containing synthetic peptides significantly improved the endothelial cell seeding of ePTFE grafts. Covalent bonding of RGD-containing peptides based on cell-adhesive regions of fibronectin, Arg-Gly-Asp-Ser (RGDS), and vitronectin, Arg-Gly-Asp-Val (RGDV) to a polyurethane graft backbone via amide bonds enhanced cell adhesion and proliferation.24 The GRGDVY-grafted substrate supported a larger number of adherent cells and a higher extent of cell spreading than the GRGDSY-grafted substrate.9 Covalent immobilization of RGD and heparin onto the surface of polymer graft improved cell retention of EC seeding.25 To further improve this material we chose to add the RGD motif which our work has shown results in excellent endothelial cell adhesion and proliferation. The RGD sequence was added as a moiety comprised of eight peptide units.21

We tested the hypothesis that the POSS–poly(carbonate-urea)urethane–RGD (POSS–PCU–RGD) octamer improves EPC cell adhesion and differentiation.

Back to Article Outline

2. Materials and Methods 

2.1. Nanocomposite synthesis of POSS–PCU–RGD 

Polymer synthesis has been described in detail elsewhere17 in brief; polycarbonate polyol and trans-cyclohexanediolisobutyl-silsesquioxane were mixed and heated to dissolve the polyhedral oligomeric silsesquioxane (POSS) cage into the polyol component before being cooled to 60°C. Methylene di-isocyanate (MDI) was added and reacted to form a pre-polymer. Dimethylacetamide (DMAC) was then added and 103mg of a nano-frame containing dendritic RGD type motif as GRGDS-LA as an octomer form reacted in at 50–60°C for 45min. A further 48g of DMAC were added slowly and the temperature adjusted to 40°C. The pre-polymer was chain-extended by the addition of diethylamine. Then 1-butanol in 10g of DMAC was added to form a 23% (w/v) solution of POSS–PCU solution with the POSS herein being at a concentration of 2%. All chemicals were purchased from Sigma Aldrich, Poole, Dorset, UK.

2.2. Peptide octamer: design and synthesis 

Originally developed as the multiple antigenic peptide (MAP) system for the production of anti-peptide antibodies, the octomer is based on a small immunogenically inert core of radially branching lysine dendrites on which a number of copies of the desired peptide is added. We have adapted the octabranching matrix made up at three levels of lysine with eight amino (Octavalent) terminals available for synthesizing eight copies of GRGDG peptide at the amino terminal of the main peptide. Glycine was introduced at the C-terminus of the main peptide to minimize any racemization during synthesis. Incorporation of this peptide format into the nanocomposite was adopted rather than pendant linkages, as this promotes stability and active conformation. Thus, two monovalent lysines were included, separated by a spacer (amino–hexanoic acid) at the C-terminus of the main peptide. The peptide was synthesized on a rink acid resin using standard solid phase methods. The peptide is synthesized as an acetylated fully protected peptide except for the 2ε-amino groups of the two lysines at the C-terminus which are deprotected during release from the solid phase. The protected peptides were 95% pure by HPLC.

2.3. Isolation of peripheral blood mononuclear cells 

The approval of the local ethical committee for this study was obtained. To obtain EPC, we took blood samples (20ml) from consented young healthy adults, mean age±SD (29.3±4.7) that had no prior stimuli to mobilise EPC from the bone marrow. Peripheral blood mononuclear cells (PBMC) were obtained using a modified density gradient technique (Lymphoprep™). Briefly, 20ml of heparinised blood was obtained from arm vein. Lymphoprep (GlaxoSmiKline, Uxbridge, UK) was added to the blood and centrifuged at 1500 RPM. PBMC were isolated and washed three times with PBS and centrifuged. The number of PBMC was counted by two independent observers, coefficient of variation 4%.

2.4. Fluorescence-activated cell sorter (FACS) analysis for CD34 

In order to determine the percentage of CD34+ cells in PBMs, the PBMC fraction was incubated with 10μl of FITC-labelled anti CD34 MAb (Miltenyi Biotec Ltd, Bisley, Surrey, UK) for 10min. PBMC also were incubated with a control phycoerythrin-labelled mouse IgG1 and FITC-labeled mouse IgG2a. Two-colour flow cytometric analyses were performed by FACS scan. Each analysis included more than 10,000 events.

2.5. Reverse transcription polymerase chain reaction (RT-PCR) analysis 

RNA was isolated from PBMC using a Quiagen RN easy mini kit following the manufacturer's instructions. Cells were homogenised using a 22 gauge needle. PCR was conducted using one step PCR (Quiagen) RT-PCR was performed on 0.5μM of RNA26 and appropriately designed primer sequence.

2.6. Peripheral blood mononuclear cells culture 

PBMs were re-suspended in 2ml of stem cell media (Sigma, UK). The PBMC fraction which contained EPC was then plated (at a density of 3×105cells/cm2) on dishes coated with POSS–PCU–RGD, POSS–PCU and uncoated dishes and cultured in the presence of vascular endothelial growth factor (VEGF; Sigma, UK) in stem cell media (Sigma, UK). Half of the media was changed every third days. Cells for immunohistochemistry were cultured on slides coated with POSS–PCU–RGD, POSS–PCU and uncoated slides at a density of 3×105cells/cm2.

2.7. Immunofluorescence for vWF, CD31, eNOS, E-selectin 

After 7 days cultured cells were immersed in a solution containing 0.2% triton X-100 in PBS for 1min, rinsed in PBS, and then incubated with normal donkey serum for 30min. Slides were incubated with the primary anti sera over night, rinsed in PBS, and mounted with vector shield containing 4′,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, Poole, Dorset, UK) and examined under a fluorescent microscope. The von Willebrand factor (vWF), CD31, E-selectin mouse antibodies were purchased from DAKO, USA. Antibodies to endothelial nitric oxide synthase (eNOS) (polyclonal, Rabbit) was purchased from Santa Cruz, CA, USA.

2.8. Alamar blue assay to assess cell viability and metabolism on nanocomposites 

AlamarBlue™ (AB) assay is a commercially available assay (Serotec Ltd, Kidlington, Oxford, UK) which has been well characterised in this laboratory as a marker of cellular viability. AB was added to cell culture media at a concentration of 10% (v/v). At day seven plates were washed with 1ml PBS and 1ml added to each well. After 4h a 100μl aliquot was removed and the absorbance at 570 and 630nm measured in a 96-well plate using a Multiscan MS UV visible spectrophotometer (Labsystems, UK).

2.9. Number of endothelial progenitor cells colonies 

At day 7 the number of EPC colonies was counted manually in each dish by two independent observers. The colonies were defined morphologically as a central core of round large cells with elongated cells at the periphery.27, 28 A central cluster of round cells alone without associated peripheral elongated cells was not counted as a colony. To confirm that colony consisted of endothelial cells indirect immunostaining of randomly selected colonies was performed with antibodies directed towards vWF, CD 31 and eNOS. The colonies were stained randomly. Results are presented as a mean of numbers of colonies formed per 106 cells plated. The cells in each experiment (in duplicate) came from the same volunteer and the same culture conditions.

Graph pad prism (version 3.0) was used to analyse data throughout. Where statistical analysis is carried out two way analysis of variance (ANOVA) was used to compare multiple groups.

Back to Article Outline

3. Results 

PBMC (1.5×107±7×105) were obtained from 20ml of venous blood. Flow cytometry indicated that 1–2% of these cells expressed CD34. A representative analysis is shown in Fig. 1. EPC are best defined as a CD133/KDR subpopulation of CD34 cells.29 To further characterise the harvested cells, mRNA expression for CD34, CD133, FlK-1/KDR and CD31 were investigate by RT-PCR. The ‘house keeping’ gene GAPDH was used as a control. Cells were shown to be positive for these markers after isolation. They remained positive at the end of the experiment (day 7) despite expressing endothelial markers at this time.

Endothelial cells colony forming units comprised clusters of round cells centrally and sprouts of spindle-shaped cells at the periphery and these began to appear between days 5 and day 7 on the POSS–PCU–RGD as shown in Fig. 2. Fewer numbers of cells became spindle shaped on POSS–PCU and uncoated dishes. In addition, fewer numbers of EPC colonies occurred on POSS–PCU and uncoated dishes as compared with POSS–PCU–RGD (Fig. 3). Colonies where identified by morphology and by staining for endothelial lineage markers as shown in Fig. 4. The endothelial phenotype was confirmed by immunostaining with antibodies specific for endothelial markers. Results are presented as a mean of numbers of colonies formed per 106 cells plated. A representative EPC preparation uniformly expressed CD31 on the cell–cell membrane borders (arrows) and expressed vWF in cytoplasmic granules (arrows), Fig. 4. In addition, cells also expressed eNOS. E-selectin was minimally expressed (data not shown). Similar results were obtained in three subsequent experiments. In each case a primary antibody free control was produced. No staining was seen in any controls (Fig. 4(d)). Cells were confirmed to be viable and metabolising by the AB assay at day 7. There were no statistical differences between the groups by AB assay as shown in Fig. 5.

  • View full-size image.
  • Fig. 4. 

    The endothelial phenotype was confirmed by immunostaining with antibodies specific for endothelial markers, (a) eNOS; (b) cell membrane and on cells border to border (arrow) CD31; (c) vWF contained within cytoplasmic granule (arrow); (d) representative control sections where nuclear staining alone is visible. All cells were counterstained with DAPI showing the nucleus as blue.

  • View full-size image.
  • Fig. 5. 

    AlamarBlueTM (AB) viability assay test on cells exposed to three group on day 7. Absorbance was measured in arbitrary units at 570nm wavelength and background at 630nm subtracted. Data are mean±SD.

Back to Article Outline

4. Discussion 

Tissue engineered cardiovascular conduits need to have the mechanical and biological properties of arteries in order to have superior patency rates. The conduits need to be non-thrombogenic, have a viscoelastic property that matches arteries and their luminal surface should be covered with a monolayer of endothelial cells. While the advantage of using EPC as a source for endothelial cells is that the procedure is much less invasive than the surgical harvest of cells from large veins and EPC have higher proliferation potential compared with mature ECs,30 these advantages can be off-set by the time and money consuming requirements to expand EPC and differentiate them into mature endothelial cells in vitro before their subsequent application.31 An alternative strategy is to prepare a material which is inherently non-thrombogenic, compliant and has the ability to promote endothelialisation from EPC directly from the blood. Previously we developed a compliant poly(carbonate-urea)urethane (PCU) grafts which offered greater degree of compliance match than either ePTFE and Dacron.32 In this study, we sought to go one step further by improving upon the luminal characteristics of PCU.

As POSS acts as an amphiphile, we synthesised a type II nanocomposite containing functional side-group POSS molecules in the form of a pendant side-chain attached to poly(carbonate-urea)urethane.33 This has resulted in a material that reduces both platelet and protein adsorption known to be responsible for activating coagulation. Our hypothesis is based on the argument that the pendant nano-cage containing silicon atoms that form foci of silicon-rich areas with increased surface free energy,34 whilst allowing the inorganic matrix to confer a ‘garter-like’ effect to its bulk to maintain its viscoelastic properties.32 The properties of this material make it an ideal candidate for further studies. The incorporation of the octameric GRGD biomolecule enhanced the ability of this amphiphilic nanocomposite to serve as a bed for stem cell attachment.

The POSS–PCU and uncoated dishes produced significantly fewer colonies than POSS–PCU–RGD. This demonstrates that the addition of the RGD moiety provided a better environment for either attachment of CD133+ cells or for their differentiation. We believe that this study demonstrates the suitability of POSS–PCU–RGD for the in situ seeding of cardiovascular devices in preference to POSS–PCU alone. RGD is not selective for EPC and in theory other cells, mainly CD14 monocytes, could attach to this surface, which might even be beneficial since studies has shown that these cells are proangiogenic and can mimic EPC.17 Previously we have shown that attachment of these other cells confers antiplatelet/antithrombogenic qualities24, 35 which provides temporary benefit during the endothelialization process. In this study, we did not attempt to have complete monolayer of differentiated endothelial cells, since exposure to flow is necessary for the differentiation process. This study showed over a short culture period EPC will adhere to this new POSS–PCU biomaterial and differentiate into cells with endothelial cell markers to indicate the potential of this technique for endothelial regeneration in situ.

In conclusion, we have developed a new bioactive nanocomposite for cardiovascular applications including the development of small diameter conduits for coronary and vascular bypass grafts and the material has been demonstrated in vitro to promote endothelialisation from circulating EPC.

Back to Article Outline

Acknowledgements 

We would like to acknowledge the financial support for development of nano materials for medical devices provided by UCL BioMedica PLC and EPSRC.

Back to Article Outline

References 

  1. Seifalian AM, Tiwari A, Hamilton G, Salacinski HJ. Improving the clinical patency of prosthetic vascular and coronary bypass grafts: the role of seeding and tissue engineering. Artif Organs. 2002;26:307–320
  2. Kannan RY, Salacinski HJ, Butler PE, Hamilton G, Seifalian AM. Current status of prosthetic bypass grafts: a review. J Biomed Mater Res B Appl Biomater. 2005;74:570–581
  3. Tiwari A, Kidane A, Salacinski HJ, Punshon G, Hamilton G, Seifalian AM. Improving endothelial cell retention for single stage seeding of prosthetic grafts: use of polymer sequences of arginine-glycine-aspartate (RGD). Eur J Vasc Endovasc Surg. 2003;25:325–329
  4. Deutsch M, Meinhart J, Fischlein T, Preiss P, Zilla P. Clinical autologous in vitro endothelialization of infrainguinal ePTFE grafts in 100 patients: a 9-year experience. Surgery. 1999;126:847–855
  5. Meinhart JG, Deutsch M, Fischlein T, Howanietz N, Froschl A, Zilla P. Clinical autologous in vitro endothelialization of 153 infrainguinal ePTFE grafts. Ann Thorac Surg. 2001;71:S327–S331
  6. Asahara T, Murohara T, Sullivan A, Silver M, van der ZR, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964–967
  7. Sales KM, Salacinski HJ, Alobaid N, Mikhail M, Balakrishnan V, Seifalian AM. Advancing vascular tissue engineering: the role of stem cell technology. Trends Biotechnol. 2005;23:461–467
  8. Campbell JH, Han CL, Campbell GR. Neointimal formation by circulating bone marrow cells. Ann N Y Acad Sci. 2001;947:18–24
  9. Adams V, Lenk K, Linke A, Lenz D, Erbs S, Sandri M, et al. Increase of circulating endothelial progenitor cells in patients with coronary artery disease after exercise-induced ischemia. Arterioscler Thromb Vasc Biol. 2004;24:684–690
  10. Vasa M, Fichtlscherer S, Adler K, Aicher A, Martin H, Zeiher AM, et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation. 2001;103:2885–2890
  11. Strehlow K, Werner N, Berweiler J, Link A, Dirnagl U, Priller J, et al. Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation. Circulation. 2003;107:3059–3065
  12. Bahlmann FH, De Groot K, Spandau JM, Landry AL, Hertel B, Duckert T, et al. Erythropoietin regulates endothelial progenitor cells. Blood. 2004;103:921–926
  13. Rafii S, Heissig B, Hattori K. Efficient mobilization and recruitment of marrow-derived endothelial and hematopoietic stem cells by adenoviral vectors expressing angiogenic factors. Gene Ther. 2002;9:631–641
  14. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 1999;5:434–438
  15. Wijelath ES, Rahman S, Murray J, Patel Y, Savidge G, Sobel M. Fibronectin promotes VEGF-induced CD34 cell differentiation into endothelial cells. J Vasc Surg. 2004;39:655–660
  16. Roberts N, Jahangiri M, Xu Q. Progenitor cells in vascular disease. J Cell Mol Med. 2005;9:583–591
  17. Rohde E, Malischnik C, Thaler D, Maierhofer T, Linkesch W, Lanzer G, et al. Blood monocytes mimic endothelial progenitor cells. Stem Cells. 2005;Sep 1 (Epub ahead of print)
  18. Havemann K, Pujol BF, Adamkiewicz J. In vitro transformation of monocytes and dendritic cells into endothelial like cells. Adv Exp Med Biol. 2003;522:47–57
  19. Alivisatos AP, Johnsson KP, Peng X, Wilson TE, Loweth CJ, Bruchez MP, et al. Organization of ‘nanocrystal molecules’ using DNA. Nature. 1996;382:609–611
  20. Kannan RY, Salacinski HJ, Butler PE, Seifalian AM. Polyhedral oligomeric silsesquioxane nanocomposites: the next generation material for biomedical applications. Acc Chem Res. 2005;38:879–884
  21. Kannan RY, Salacinski HJ, Odlyha M, Butler PE, Seifalian AM. The degradative resistance of polyhedral oligomeric silsesquioxane nanocore integrated polyurethanes: an in vitro study. Biomaterials. 2006;27:1971–1979
  22. Salacinski HJ, Handcock S, Seifalian AM. Polymer for use in conduits, medical devices and biomedical surface modification. International Patent Application No 2005; PCT/GB2005/000189.
  23. Rashid ST, Salacinski HJ, Button MJ, Fuller B, Hamilton G, Seifalian AM. Cellular engineering of conduits for coronary and lower limb bypass surgery: role of cell attachment peptides and pre-conditioning in optimising smooth muscle cells (SMC) adherence to compliant poly(carbonate-urea)urethane (MyoLink) scaffolds. Eur J Vasc Endovasc Surg. 2004;27:608–616
  24. Kidane AG, Salacinski HJ, Punshon G, Ramesh B, Srai KS, Seifalian AM. Synthesis and evaluation of amphiphilic RGD derivatives: uses for solvent casting in polymers and tissue engineering applications. Med Biol Eng Comput. 2003;41:740–745
  25. Tiwari A, Salacinski HJ, Punshon G, Hamilton G, Seifalian AM. Development of a hybrid cardiovascular graft using a tissue engineering approach. FASEB J. 2002;16:791–796
  26. Vara DS, Punshon G, Sales KM, Salacinski HJ, Dijk S, Brown RA, et al. Development of an RNA isolation procedure for the characterisation of human endothelial cell interactions with polyurethane cardiovascular bypass grafts. Biomaterials. 2005;26:3987–3993
  27. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003;348:593–600
  28. Lev EI, Kleiman NS, Birnbaum Y, Harris D, Korbling M, Estrov Z. Circulating endothelial progenitor cells and coronary collaterals in patients with non-ST segment elevation myocardial infarction. J Vasc Res. 2005;42:408–414
  29. Wu X, Rabkin-Aikawa E, Guleserian KJ, Perry TE, Masuda Y, Sutherland FW, et al. Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells. Am J Physiol Heart Circ Physiol. 2004;287:H480–H487
  30. Zisch AH. Tissue engineering of angiogenesis with autologous endothelial progenitor cells. Curr Opin Biotechnol. 2004;15:424–429
  31. Mooney D. Nimble progenitors rescue vascular grafts. Nat Med. 2001;7:996–997
  32. Tai NR, Salacinski H, Edwards A, Hamilton G, Seifalian AM. Compliance properties of conduits used in vascular reconstruction. Br J Surg. 2000;87:1480–1488
  33. Seifalian AM, Salacinski HJ, Tiwari A, Edwards A, Bowald S, Hamilton G. In vivo biostability of a poly(carbonate-urea)urethane graft. Biomaterials. 2003;24:2549–2557
  34. Silver JH, Lin JC, Lim F, Tegoulia VA, Chaudhury MK, Cooper SL. Surface properties and hemocompatibility of alkyl-siloxane monolayers supported on silicone rubber: effect of alkyl chain length and ionic functionality. Biomaterials. 1999;20:1533–1543
  35. Kidane AG, Salacinski H, Tiwari A, Bruckdorfer KR, Seifalian AM. Anticoagulant and antiplatelet agents: their clinical and device application(s) together with usages to engineer surfaces. Biomacromolecules. 2004;5:798–813

☆ Has been awarded The Young Vascular Surgeons' prize at ESVS meeting Helsinki 2005.

PII: S1078-5884(06)00003-7

doi:10.1016/j.ejvs.2005.11.034

European Journal of Vascular & Endovascular Surgery
Volume 32, Issue 1 , Pages 76-83, July 2006

Article Tools

* Image Usage & Resolution