John Paul Kirton

Vascular repair by endothelial progenitor cells

Accumulating evidence indicates the impact of endothelial progenitor cells (EPCs) in vascular repair. In patients, the number of EPCs is negatively correlated with the severity of atherosclerosis. In various animal models, transplantation of bone marrow-derived progenitor cells could sufficiently rescue organ function and enhance vascular repair and tissue regeneration. Increase in the number of circulating progenitors, induced by cell transfusion or enhanced mobilization, can also enhance restoration and integrity of the endothelial lining, suppress neointimal formation, and increase blood flow to ischaemic sites. However, the beneficial outcome of EPC infusion very much depends on the growth and differentiation factors within the tissue, cell-to-cell interactions, and the degree of injury. As highlighted by several studies, EPCs derive from different sources including bone marrow and non-bone marrow organs such as the spleen, the functional repair properties of which may vary with the maturation state of the cell. Thus, understanding the molecular mechanisms involved in EPC-repairing processes is essential. In the present review we focus on the role of EPCs in vascular diseases, and we provide an update on the mechanisms of EPC mobilization, homing, and differentiation.

Endothelial precursors in vascular repair

The endothelium is an essential component of the cardiovascular system, playing a vital role in blood vessel formation, vascular homeostasis, permeability and the regulation of inflammation. The integrity of the endothelial monolayer is also critical in the prevention of atherogenesis and as such, restoration of the monolayer is essential following damage or cell death. Over the past decade, data has suggested that progenitor cells from different origins within the body are released into the circulation and contribute to re-endothelialisation. These cells, termed endothelial progenitor cells (EPCs), also gave rise to the theory of new vessel formation within adults (vasculogenesis) without proliferation and migration of mature endothelial cells (angiogenesis). As such, intense research has been carried out identifying how these cells may be mobilised and contribute to vascular repair, either encouraging vasculogenesis into regions of ischemia or the re-endothelialisation of vessels with a dysfunctional endothelium. However, classification and isolation procedures have been a major problem in this area of research and beneficial use for therapeutic application has been controversial. In the present review we focus on the role of EPCs in vascular repair. We also provide an update on EPC classification and discuss autologous stem cell-derived endothelial cell (EC) as a functional source for therapy.

Contribution of stem cells to neointimal formation of decellularized vessel grafts in a novel mouse model.

Artificial vessel grafts are often used for the treatment of occluded blood vessels, but neointimal lesions commonly occur. To both elucidate and quantify which cell types contribute to the developing neointima, we established a novel mouse model of restenosis by grafting a decellularized vessel to the carotid artery. Typically, the graft developed neointimal lesions after 2 weeks, resulting in lumen closure within 4 weeks. Immunohistochemical staining revealed the presence of endothelial and smooth muscle cells, monocytes, and stem/progenitor cells at 2 weeks after implantation. Explanted cultures of neointimal tissues displayed heterogeneous outgrowth in stem cell medium. These lesional cells expressed a panel of stem/progenitor markers, including c-kit, stem cell antigen-1 (Sca-1), and CD34. Furthermore, these cells showed clonogenic and multilineage differentiation capacities. Isolated Sca-1(+) cells were able to differentiate into endothelial and smooth muscle cells in response to vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF)-BB stimulation in vitro. In vivo, local application of VEGF to the adventitial side of the decellularized vessel increased re-endothelialization and reduced neointimal formation in samples at 4 weeks after implantation. A population of stem/progenitor cells exists within developing neointima, which displays the ability to differentiate into both endothelial and smooth muscle cells and can contribute to restenosis. Our findings also indicate that drugs or cytokines that direct cell differentiation toward an endothelial lineage may be effective tools in the prevention or delay of restenosis.

c-Kit+ progenitors generate vascular cells for tissue-engineered grafts through modulation of the Wnt/Klf4 pathway

The development of decellularised scaffolds for small diameter vascular grafts is hampered by their limited patency, due to the lack of luminal cell coverage by endothelial cells (EC) and to the low tone of the vessel due to absence of a contractile smooth muscle cells (SMC). In this study, we identify a population of vascular progenitor c-Kit+/Sca-1- cells available in large numbers and derived from immuno-privileged embryonic stem cells (ESCs). We also define an efficient and controlled differentiation protocol yielding fully to differentiated ECs and SMCs in sufficient numbers to allow the repopulation of a tissue engineered vascular graft. When seeded ex vivo on a decellularised vessel, c-Kit+/Sca-1-derived cells recapitulated the native vessel structure and upon in vivo implantation in the mouse, markedly reduced neointima formation and mortality, restoring functional vascularisation. We showed that Krüppel-like transcription factor 4 (Klf4) regulates the choice of differentiation pathway of these cells through β-catenin activation and was itself regulated by the canonical Wnt pathway activator lithium chloride. Our data show that ESC-derived c-Kit+/Sca-1-cells can be differentiated through a Klf4/β-catenin dependent pathway and are a suitable source of vascular progenitors for the creation of superior tissue-engineered vessels from decellularised scaffolds.

Cells and Vascular Tissue Engineering

Cardiovascular disease is the leading cause of death worldwide. As such, vascular reconstruction and graft bypass surgery are in high demand to slow the rate of morbidity following the onset of this pathology. However, the use of non-biocompatible synthetic grafts, especially those of small diameter (6 mm or less), frequently leads to thrombosis and occlusion of the vessel. The field of vascular tissue engineering provides an alternative method to generate small (and large)-diameter bypass grafts that can support cell growth and are expected to exhibit long-term patency. Following the generation of the first in vitro blood vessel over 30 years ago, there has been considerable progress in this area in terms of scaffold availability, construction of the vessels and application of stem cells. This chapter will specifically focus on the use of stem cells in the generation of vascular grafts. Here we will highlight the current scaffolds available for seeding cells, the alternate stem cell sources and their isolation, the methods used to differentiate stem cells into vascular lineages and their application in generating blood vessels in vitro. The future hurdles that must be overcome before tissue-engineered blood vessels can be applied to a clinical setting will also be discussed.

21 Proteomic analysis of iPS and embryonic stem cells identifies alternate vascular cell differentiation properties

The generation of induced pluripotent stem (iPS) cells from somatic cells can be a useful tool for regenerative medicine. The artificial nature of iPS cells raises concerns whether iPS and embryonic stem (ES) cells, are molecularly and functionally comparable. In this study we generated iPS cells using a genomic integration free method, and using the advance of proteomics, we aimed to elucidate any differences in the protein profile between iPS and ES cells, in terms of pluripotency and differentiation potential to vascular cell lineages. The results demonstrated that 180 proteins were differentially expressed, in which 66% showed a decreased expression pattern in iPS cells. Functional classification analysis revealed that these proteins were associated with mRNA processing, energy metabolism, cytoskeleton, proliferation, and differentiation. Further experiments demonstrated that iPS cells have a decreased proliferation capacity when compared to ES cells. Importantly, iPS cells also displayed a greater potential to differentiate into smooth muscle and endothelial cell lineages, when compared to ES cells. When iPS and ES cells were seeded on collagen IV-coated dishes and cultured with differentiation media, MEM supplemented with 10% serum, iPS cells displayed a differentiation morphology as early as day 3 in comparison to ES cells, and real-time PCR data confirmed that iPS express smooth muscle cell markers such as SMA, calponin, and SM22 in higher levels. When vascular endothelial growth factor (VEGF) was added to differentiation media, iPS cells expressed endothelial markers such as CD31, CD144, Flk-1 and Flt-1 as early as day 3 on differentiation, while ES cells showed limited expression of these markers during the early stages of differentiation. Therefore, identifying differentially regulated protein expression between the two cell types and the pathways they influence, will allow us to elucidate the mechanisms that lead to vascular cell differentiation, thus making iPS technology a successful tool for regenerative medicine and treatment of vascular diseases.