Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

75.M. S. Aymerich, E. M. Alberdi, A. Martinez, and S. P. Becerra, Evidence for pigment epithelium-derived factor receptors in the neural retina. Invest. Ophthalmol. Vis. Sci. 42, 3287-3293, (2001).

76.T. Yabe, D. Wilson, and J. P. Schwartz, NFkappaB activation is required for the neuroprotective effects of pigment epithelium-derived factor (PEDF) on cerebellar granule neurons. J. Biol. Chem. 276, 43313-43319, (2001).

77.T. Yabe, J. T. Herbert, A. Takanohashi, and J. P. Schwartz, Treatment of cerebellar granule cell neurons with the neurotrophic factor pigment epithelium-derived factor in vitro enhances expression of other neurotrophic factors as well as cytokines and chemokines. J. Neurosci. Res. 77, 642-652, (2004).

78.O. V. Volpert, T. Zaichuk, W. Zhou, F. Reiher, T. A. Ferguson, P. M. Stuart, M. Amin, and N. P. Bouck, Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor. Nat. Med. 8, 349-357, (2002).

79.M. Oukka, I. C. Ho, F. C. de la Brousse, T. Hoey, M. J. Grusby, and L. H. Glimcher, The transcription factor NFAT4 is involved in the generation and survival of T cells. Immunity 9, 295-304, (1998).

80.M. Maden, Retinoid signalling in the development of the central nervous system. Nat. Rev. Neurosci. 3, 843-853, (2002).

81.J. Tombran-Tink, N. Lara, S. E. Apricio, P. Potluri, S. Gee, J. X. Ma, G. Chader, and

C.J. Barnstable, Retinoic acid and dexamethasone regulate the expression of PEDF in retinal and endothelial cells. Exp. Eye Res. 78, 945-955, (2004).

82.W. R. Lo, L. L. Rowlette, M. Caballero, P. Yang, M. R. Hernandez, and T. Borras, Tissue differential microarray analysis of dexamethasone induction reveals potential mechanisms of steroid glaucoma. Invest. Ophthalmol. Vis. Sci. 44, 473-485, (2003).

83.S. Yamagishi, Y. Inagaki, S. Amano, T. Okamoto, and M. Takeuchi, Up-regulation of vascular endothelial growth factor and down-regulation of pigment epithelium-derived factor messenger ribonucleic acid levels in leptin-exposed cultured retinal pericytes. Int.

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84.E. M. Alberdi, J. E. Weldon, and S. P. Becerra, Glycosaminoglycans in human retinoblastoma cells: heparan sulfate, a modulator of the pigment epithelium-derived factor-receptor interactions. BMC Biochem. 4, 1, (2003).

85.N. Yasui, T. Mori, D. Morito, O. Matsushita, H. Kourai, K. Nagata, and T. Koide, Dual-site recognition of different extracellular matrix components by anti-angiogenic/neurotrophic serpin, PEDF. Biochemistry 42, 3160-3167, (2003).

86.L. Notari, A. Miller, A. Martinez, J. Amaral, M. Ju, G. Robinson, L. E. Smith, and

S.P. Becerra, Pigment epithelium-derived factor is a substrate for matrix metalloproteinase type 2 and type 9: implications for downregulation in hypoxia. Invest. Ophthalmol. Vis. Sci. 46, 2736-2747, (2005).

87.J. M. Sivak and M. E. Fini, MMPs in the eye: emerging roles for matrix metalloproteinases in ocular physiology. Prog. Retin. Eye Res. 21, 1-14, (2002).

88.C. Shao, J. Sima, S. X. Zhang, J. Jin, P. Reinach, Z. Wang, J. X. Ma, Suppression of corneal neovascularization by PEDF release from human amniotic membranes. Invest. Ophthalmol. Vis. Sci. 45, 1758-1762, (2004).

89.J. Amaral, B. Burkam, and P. Becerra, Antiangiogenic Effects of PEDF Peptide fragments and Cleaved PEDF. Invest. Ophthalmol. Vis. Sci. 46, ARVO E-Abstract 453, (2005).

90.H. Liu, J. G. Ren, W. L. Cooper, C. E. Hawkins, M. R. Cowan, and P. Y. Tong, Identification of the antivasopermeability effect of pigment epithelium-derived factor and its active site. Proc. Natl. Acad. Sci. USA 101, 6605-6610, (2004).

91.E. Chaum and M. P. Hatton, Gene therapy for genetic and acquired retinal diseases.

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92.P. A. Campochiaro, Gene therapy for retinal and choroidal diseases. Expert Opin. Biol. Ther. 2, 537-544, (2002).

93.A. Wenzel, T. A. Afanasieva, M. W. Seeliger, C. Grimm, M. Samardzija, S. Hotop, and

C.E. Remé, Balance of PEDF and VEGF controls light induced photoreceptor apoptosis. Invest. Ophthalmol. Vis. Sci. 45, ARVO E-Abstract 779, (2004).

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C.Cingolani, H. Lai, L. Wei, and P. A. Campochiaro, Periocular injection of an adenoviral vector encoding pigment epithelium-derived factor inhibits choroidal neovascularization. Gene Ther. 10, 637-646, (2003).

95.P. Gehlbach, A. M. Demetriades, S. Yamamoto, T. Deering, W. H. Xiao, E. J. Duh,

H.S. Yang, H. Lai, I. Kovesdi, M. Carrion, L. Wei, and P. A. Campochiaro, Periocular gene transfer of sFlt-1 suppresses ocular neovascularization and vascular endothelial growth factor-induced breakdown of the blood-retinal barrier. Hum. Gene Ther. 14, 129-141, (2003).

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B.Harris, M. Hamilton, L. Wei, and P. A. Campochiaro, Periocular gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization in a humansized eye. Hum. Gene Ther. 16, 473-478, (2005).

97.K. Mori, P. Gehlbach, A. Ando, D. McVey, L. Wei, and P. A. Campochiaro, Regression of ocular neovascularization in response to increased expression of pigment epitheliumderived factor. Invest. Ophthalmol. Vis. Sci. 43, 2428-2434, (2002).

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Q.D. Nguyen, and J. U. Sung, Clinical protocol. An open-label, phase I, single administration, dose-escalation study of ADGVPEDF.11D (ADPEDF) in neovascular age-related macular degeneration (AMD). Hum. Gene Ther. 12, 2029-2032, (2001).

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100.I. Semkova, F. Kreppel, G. Welsandt, T. Luther, J. Kozlowski, H. Janicki, S. Kochanek, and U. Schraermeyer, Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye. Proc. Natl. Acad. Sci. USA 99, 13090-13095, (2002).

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Chapter 18

CIRCULATING ENDOTHELIAL PROGENITOR CELLS AND ADULT VASCULOGENESIS

Sergio Caballero, Nilanjana Sengupta, Lynn C. Shaw, and Maria B. Grant

Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida

Abstract: Postnatal neovascularization (NV) has previously been considered synonymous with proliferation and migration of pre-existing, fully differentiated endothelial cells resident within parent vessels. The finding that circulating endothelial progenitor cells (EPCs) may home to sites of NV and differentiate in situ is consistent with vasculogenesis, a critical paradigm for the establishment of vascular networks in the embryo. While the percent contributions of angiogenesis and vasculogenesis to postnatal NV remain to be clarified, our observations in the eye, together with recent reports from other investigators in other tissues and pathologies, support that growth and development of new blood vessels in the adult are not restricted to angiogenesis but encompass both embryonic and adult mechanisms. As a corollary, augmented or retarded NV, whether endogenous or iatrogenic, likely includes enhancement or impairment of vasculogenesis. In this chapter, the molecular and cellular factors that play a role in EPC involvement in NV are discussed.

1.INTRODUCTION

The endothelium is the single-cell lining covering the internal surface of blood vessels, cardiac valves, and numerous body cavities. Its roles include prevention of thrombosis, leukocyte and platelet adhesion, and vessel wall contraction and relaxation.1 Endothelial dysfunction predisposes a person to hypertension, thrombosis, atherosclerosis, and diabetic microand macrovascular complications.2 Endothelial progenitor cells (EPCs) play an important role in maintenance of endothelial cell health, being important in both re-endothelialization and neovascularization (NV).

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In 1991, George et al. unequivocally demonstrated the presence of circulating endothelial cells (CECs) in whole blood using an endothelial cellspecific antibody.3 Since that time, a number of different laboratories have identified CECs in whole blood in a variety of pathological conditions by the use of endothelial cell-specific monoclonal antibodies. In normal individuals, there are approximately zero to 20 CECs per milliliter of blood. CECs may be derived from two sources: the peripheral vasculature or, more interestingly, the bone marrow. Cells derived from the peripheral vasculature are mature endothelial cells “shed” into the circulation; they express phenotypic endothelial cell markers such as von Willebrand factor, VEcadherin, CD146, or TE-7. These mature endothelial cells may detach due to mechanical disruption or as a result of apoptosis, although TUNEL assays for apoptosis performed on CECs were negative.4,5 If CECs originate from the bone marrow, they are derived from EPCs and can fully differentiate into endothelial cells, expressing mature endothelial cell markers. This chapter focuses on bone marrow-derived EPCs.

2.DEFINING THE EPC

The unequivocal identification of EPCs is difficult due to their paucity of surface markers as well as their variable phenotype. The lineage and exact phenotype of EPCs are still inadequately characterized. In the simplest terms, EPCs are cells that possess the ability to mature into the cells that line the lumen of blood vessels.6 The first evidence indicating the presence of EPCs in the adult circulation emerged when mononuclear blood cells from healthy human volunteers were shown to acquire an endothelial cell-like phenotype in vitro and to incorporate into capillaries in vivo.7 Because both CD34 and vascular endothelial growth factor receptor type 2 (VEGFR-2) are expressed on mature endothelial cells as well as EPC, a search for more unique EPC markers continues. CD34+ cells express the early hematopoietic progenitor cell marker CD133 (AC133), which is not expressed after differentiation.8 Using CD133 expression to define a very early subset of progenitor cells, Peichev et al. isolated a CD133+/CD34+/VEGFR-2+ subpopulation of cells able to differentiate into mature endothelial cells.9 After seven days of culture on fibronectin, CD34+ mononuclear cells display an endothelial cell phenotype, incorporate acetylated low density lipoproteins, produce nitric oxide when stimulated with VEGF, and express PECAM and Tie-2 receptor.7

CD133+ hematopoietic progenitor cells comprise the more immature subset of CD34+ cells, and these cells can repopulate sheep bone marrow.10 Some investigators believe that a unique subset of cells expressing CD133,

CD34, and VEGFR-2 is the primary source of EPCs.9 CD133+/CD34+ cells are thought to be more primitive EPCs, whereas CD133-/CD34+/VEGFR-2+ cells may represent a more mature, differentiated population of endothelial precursor cells.9,11 In support of this, CD133+/CD34+ cells do not express VE-cadherin or von Willebrand factor, and only 3% of these cells express VEGFR-2. However, after three weeks of culture and further purification with Ulex europaeus agglutinin (a lectin specific for endothelial cells), these cells expressed several endothelial markers including von Willebrand factor, CD146, CD105, E-selectin, VCAM-1, and VE-cadherin.12

Although it is not yet clear what markers precisely define an EPC, it is clear that these cells derived from the bone marrow will populate an area of neoangiogenesis. Eight to 11% of the endothelial cells in a neovascular mouse model are of EPC origin, whereas hematopoietic progenitors do not populate the vasculature in stable uninjured adult tissue.13 Similar results are seen in the NV that occurs in the endometrium during ovulation and wound healing in mice.14 Not only are there EPCs in the circulation that have the capacity to populate neovasculature, but there are also circulating hematopoietic stem cells (HSCs) that can repopulate the bone marrow.15

3.THE ROLE OF ADULT EPCs

EPC mobilization resulting in re-endothelialization was first characterized by studies in which adult dogs underwent bone marrow (BM) transplantation and thoracic aorta implantation of Dacron grafts. After three months, the grafts were removed and found to be colonized with CD34+ endothelial cells of donor origin. This novel observation suggested that endothelialization arose from BM-mobilized EPCs rather than resident endothelium on preexisting vessels.16 The surfaces of left ventricular assist devices were found to be colonized with CD133+/VEGFR-2+ cells.9 Together, these studies suggested the existence of a population of EPCs in the peripheral circulation that contributes to rapid endothelialization (Figure 1). EPCs modulate reendothelialization at sites of endothelial cell damage.8,17 Following carotid artery endothelial injury, BM-derived endothelial cells were observed at the site of injury.17 These studies suggest that EPCs participate in the maintenance of vascular homeostasis by restoring the endothelium.

Figure 18-1. EPCs facilitate NV and angiogenesis. Mobilization of EPCs from BM is enhanced by growth factors and cytokines including VEGF, SDF-1, GM-CSF, and pharmacological agents such as statins. EPCs participate with resident endothelial cells in both physiological (beneficial) NV such as wound healing, revascularization following myocardial infarction, and in reperfusion of ischemic limbs and in pathological NV such as in preretinal NV in diabetic individuals.

3.1The Role of EPCs in Neovascularization

The traditional view of adult vascularization holds that new blood vessels form by angiogenesis, the sprouting of new vessels from existing vessels. This process relies on proliferation, migration, and remodeling of fully differentiated resident endothelial cells.18 In contrast, vasculogenesis represents the formation of blood vessels de novo from primitive EPCs during embryological development.19 With the discovery of circulating

EPCs, the current view holds that a combination of angiogenesis and vasculogenesis contributes to NV in the adult (Figure 1). EPC-dependent NV in adults is a means of blood vessel formation, paralleling developmental vasculogenesis in the embryo. EPCs may serve as the substrate for new vessel formation and simultaneously exert a paracrine effect to promote angiogenesis. Factors such as VEGF, angiopoietin, basic fibroblast growth factor (FGF-2), granulocyte-macrophage colony-stimulating factor (GMCSF), matrix metalloprotease-9 (MMP-9), and pharmacological agents such as statins influence the process of EPC incorporation.8

4.FACTORS INFLUENCING EPC MOBILIZATION

EPCs reside in the BM in close association with HSCs and BM stromal cells that provide the microenvironment for hematopoiesis.6 Under steady-state physiological conditions, EPCs represent only 0.01% of circulating mononuclear cells.20 In order for the progenitor cells to leave the BM and mobilize, they must first lose contact with the stromal cells. Endogenous stimuli (such as tissue ischemia) and exogenous effectors (such as cytokine administration) have been shown to mobilize EPCs (Figure 2). Regional ischemia results in an increase in the number of circulating EPCs.21 Patients with vascular trauma, acute myocardial infarction (AMI), or diabetic retinopathy have increased mobilization of EPCs.21 EPC numbers increase in AMI patients compared to control subjects, and the increase correlates to elevated plasma levels of VEGF in these patients.22 The regenerative capacity of EPCs may thus be partially mediated by growth factor and chemokine release.

VEGF promotes mobilization of EPCs and their incorporation into sites of NV.23,24 It promotes angiogenesis by inducing proliferation, differentiation, and chemotaxis of endothelial cells.25 It is essential for hematopoiesis and angiogenesis, as illustrated by the death of mice in utero that have a single VEGF allele.26 Multiple isoforms of VEGF are secreted – VEGF165 being the most abundant – that exert their biological effects through interaction with two tyrosine kinase receptors, VEGFR-1 and VEGFR-2.27 Both of these receptors are expressed on HSCs and EPCs. The secretion of VEGF by cells in the BM compartment induces EPC proliferation, BM remodeling, adhesion molecule expression, and EPC migration from the BM.20 After VEGF administration in rodents, circulating EPCs were increased in the circulation and displayed enhanced proliferative and migratory activity.14 In patients with lower limb ischemia who received VEGF gene transfer, there was an overall increase in circulating EPCs by more than two-fold.24 These results suggest that VEGF overexpression can

mobilize EPCs in humans. In addition to VEGF, other angiogenic growth factors, including angiopoietin-1, FGF-2, and stromal cell-derived growth factor-1 (SDF-1), stimulate EPC mobilization and recruitment.23,28

Figure 18-2. Ischemic tissue (here depicted as retina, but could be heart or any tissue) sends out signals (VEGF, PLGF, SDF-1), which results in mobilization of HSCs and EPCs from the bone marrow cavity. These factors stimulate the expression of NO, which leads to production of proMMP-9, which becomes activated and results in release of membrane bound kit ligand (mKitL). mKitL becomes soluble kit ligand (KitL), which facilitates the release of progenitor cells from their “niche” and mobilizes them into the circulation via the bone marrow sinusoids. Once in the circulation, these cells are recruited to areas of ischemia by the local signals, VEGF and SDF-1. NO acts at multiple steps in this process including increased expression of MMP-9 in bone marrow stromal cells and increasing HSC and EPC migratory ability, both while in the bone marrow and in the circulation.

Several chemokines and cytokines are also able to promote the mobilization of EPCs. Placental growth factor (PlGF), a member of the VEGF family, was shown to stimulate collateral vessel formation in

ischemic heart and limbs by acting via VEGFR-1 and inducing the recruitment of BM-derived EPCs.23,29 VEGF can also induce the release of

GM-CSF by BM endothelial cells.30 Exogenously administered GM-CSF specifically mobilizes EPCs in mice and contributes to improved vascularization in ischemic hind limbs.21 GM-CSF administered to coronary artery disease patients demonstrated an improvement in myocardial collateral flow.30 While not measured specifically, EPC mobilization and recruitment to the myocardium may have contributed to the favorable outcome. Granulocyte colony stimulating factor (G-CSF), like GM-CSF, has been shown to increase the number of CD34+ cells that could stimulate NV in regions of ischemic myocardium.31 The chemokine stem cell factor (SCF, sKitL) also mobilizes stem cells.8

The recruitment of progenitor cells from the BM also appears to require MMP-9 activity.32 MMP-9-/- mice have impaired stem cell mobilization, and MMP inhibitors effectively block EPC mobilization. MMP-9 activity causes the shedding of SCF, which favors recruitment of c-kit+ progenitors, including EPCs, from the BM. Metalloproteinase activity promotes the motility of otherwise quiescent EPCs by releasing stem cell-active cytokines such as SCF from the BM stroma. PlGF released from ischemic tissue recruits BM-derived progenitors by upregulating MMP-9 activity and meditating SCF release.33 Interestingly, the same factors responsible for mobilization also influence EPC migration and incorporation. We and others have shown that EPC recruitment correlates directly with VEGF levels,

whether by endogenous expression from injured tissue or by exogenous delivery of VEGF.20,34

4.1SDF-1 and CXCR4 in EPC Recruitment

Chemokines are a group of structurally related proteins that participate extensively in mechanisms of leukocyte trafficking. These molecules are involved in aspects of the immune system related to immune surveillance, innate and adaptive immunity, and inflammation.35 Functional roles for chemokine peptides extend beyond mechanisms involved in chemoattraction; other fundamental physiological processes also appear to be regulated by chemokines. Chemokines exert their biological effects by binding and activating receptors in the G-protein coupled receptor (GPCR) superfamily. To date, nearly 50 chemokine peptides are known, while the number of chemokine receptors that have been characterized has reached 19; each gene family is likely to see further expansion. Chemokines usually

interact with their receptors in a specific manner, although some chemokines bind several receptors and some receptors bind multiple chemokines. Chemokine receptors are expressed by a variety of cell types, classically characterized on the vast array of leukocytic cell populations. These receptors regulate a number of cellular signaling pathways in a chemokinedependent manner.

SDF-1 is a member of the CXC chemokine subfamily that was initially identified from a signal sequence trap cloning strategy.36 The SDF-1 gene has at least two distinct transcripts encoding proteins that differ in their C- termini; sequences encoding the SDF-1 protein are highly conserved across species. SDF-1 was initially identified as a BM stromal cell-derived chemoattractant for hematopoietic progenitor (CD34+) cells.37 In 1996, two groups independently reported that SDF-1 is a ligand for a previously identified orphan GPCR termed fusin/LESTR/HUMSTR.38,39 The receptor was subsequently identified as an SDF-1 binding protein, and SDF-1- dependent signaling through this receptor was established. The receptor was given the name CXCR4. The significance of this receptor was enhanced when it was discovered to be one of two major co-receptors for HIV-1.40

Roles for SDF-1 and CXCR4 in development are indicated from studies of SDF-1 and CXCR4 gene-disrupted mice. Animals with targeted deletions in either the SDF-1 or CXCR4 genes display similar phenotypes, dying perinatally, presumably due to specific effects on organogenesis.41,42 The mutant mice exhibit defects in B-cell lymphopoiesis, BM myelopoiesis, and cardiac septal formation. Endothelial cells in developing vascular beds express CXCR4, and animals deficient in either SDF-1 or CXCR4 do not form large vessels within the gastrointestinal tract, clearly indicating a role for this chemokine ligand/receptor pair in vascular development. CD34+ EPCs express functional CXCR4, migrating in response to SDF-1.9 CXCR4 expression by endothelial cells of various origins is also well documented.43 In endothelial cells, CXCR4 expression is increased after treatment with VEGF or FGF-2.44 SDF-1 has also been shown to stimulate VEGF expression in a number of cells.45

One common element in the different environments where vasculogenesis is believed to occur is the presence of a hypoxic stimulus. Ceradini et al. identified SDF-1 and CXCR4 as critical mediators for the ischemia-specific recruitment of circulating progenitor cells.46 They found that endothelial expression of SDF-1 acts as a signal indicating the presence of tissue ischemia, and that its expression is directly regulated by hypoxiainducible factor-1 (HIF-1). SDF-1 is the only chemokine known to be regulated in this manner. Proliferation, patterning, and assembly of recruited progenitors into functional blood vessels are also influenced by tissue oxygen tension and hypoxia. Both SDF-1 and hypoxia are present in the BM