Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

33 ºC, but became quiescent within 2 days when shifted to 37 ºC. Several

immortalized brain capillary endothelial cell lines also have been established from transgenic mice harboring the SV-40 Tag.54,55

Reversion of immortalization seems to be necessary in some experimental models where the immortalizing gene may influence the experimental outcome.56 In H-2Kb-tsA58 transgenic mice, the temperature sensitive SV-40 Tag gene is controlled by the H-2k(b) class-I histocompatibility promoter, which is inducible by INF-gamma. Using this mouse, Lindington and co-workers established a cardiac endothelial cell line, which grew exponentially at the permissive temperature and in the presence of INF-gamma.57 At 38 ºC and in the absence of INF-gamma, the cells stopped growing and became responsive to basic FGF, VEGF, and EGF. By cross-breeding this same mouse with the uPAR-/- mouse, we have been able to isolate uPAR-/- brain endothelial cell lines; characterization of these cells is underway in our laboratory (Behzadian et al., unpublished). Urokinase plasminogen activator and its receptor (uPA/uPAR) have been implicated in the regulation of endothelial barrier function and endothelial cell migration. Isolation of mutant endothelial cells from transgenic uPAR-/- mice provides an important in vitro model for studying the function of the uPA/uPAR system in retinopathy.

1.4Endothelial Precursor Cells

The process of angiogenesis, defined as formation of new blood vessels by sprouting from pre-existing blood vessels, has been classically distinguished from vasculogenesis, which is the mobilization and assembly of mesenchimal endothelial precursor cells into vascular structures. This distinction was based not only on differences in the way the two processes occur, but also on the notion that vessel growth during embryonic development mainly involves vasculogenesis, whereas postnatal neovascularization occurs mainly by angiogenesis. However, recent evidence indicates that vasculogenesis plays a significant role in postnatal neovascularization.58-60 The identification of endothelial precursor cells in the adult bone marrow and circulating blood61 led to the discovery of endothelial precursor cells in sites of neovascularization and changed our understanding of postnatal vessel growth and repair.62 Active recruitment of endothelial precursor cells has been demonstrated in the ischemic retina,63-67 suggesting that therapies targeting these precursor cells will help in blocking pathological neovascularization in retina.

Studies of bone marrow-derived hematopoietic stem cells (HSCs) and endothelial progenitor cells are thoroughly discussed elsewhere in this volume. Our focus is on potential use of endothelial precursor cells for in

vitro studies. Using cultured precursor cells, one can study the effects of chemokines, pharmaceutical reagents, and cellular mediators on precursor cell attachment, migration, differentiation, and resistance to stress conditions. This information will contribute to our understanding of the biology of precursor cells as well as their potential therapeutic use. Furthermore, genetic manipulation of endothelial precursor cells, as achieved by gene transduction, can be used to selectively promote the expression of proor anti-angiogenic factors at sites of tissue injury.68 Another important application for precursor cell cultures is to find new ways of enhancing their growth rate in order to obtain larger numbers of cells for transplantation in conditions where vascular growth is needed.

Sources of endothelial precursor cells include bone marrow explants, umbilical cord, and peripheral blood. The cells can be isolated from the mononuclear cell fraction of peripheral blood by density gradients and then identified based on the expression of specific surface antigens including CD34, VEGF receptor-2 (VEGFR-2, Flk-1), and the orphan receptor AC133.69,70 This particular pattern of surface antigens is also present on HSCs, demonstrating that endothelial precursor cells are related to HSCs, with which they share a common progenitor, the hemangioblast.71 The expression of surface antigens is strictly dependent on the stage of endothelial precursor cell differentiation. For example, immature circulating endothelial precursor cells express AC133, but this antigen is not found on the surface of more mature “committed” endothelial precursor cells.72 The phenotypic switch of endothelial precursor cells to the mature, terminally differentiated endothelial cell phenotype can be monitored by the appearance of other specific surface antigens such as the von Willebrand factor.73

Endothelial precursor cells are specifically sensitive to bioactive peptides, including VEGF, insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF), and an appropriate balance of different cytokines is critical for preventing their differentiation into mature endothelial cells. The maintenance of their undifferentiated state in vitro is essential to the preservation of their self-renewal abilities and stem cell-like properties. Specific genetic analysis of endothelial precursor cells has identified characteristic profiles of gene expression common to many stem cells; these are thought to be involved in the maintenance of their so-called “stemness.”74,75

Cultured endothelial precursor cells have been used in ex vivo models of hind limb and myocardial ischemia.59 Recent studies using these models have shown that endothelial precursor cells can display many of the morphological and functional characteristics of mature endothelial cells, such as formation of vascular-like structures in Matrigel.76 However their

replicative capacity and their stress resistance are greater than mature endothelial cells. Endothelial precursor cells also appear to reach senescence at a much slower pace than endothelial cells, and this effect has been

explained by enhanced antioxidant abilities as well as reduced telomerase activity in these cells.77,78

Drugs’ effects on endothelial precursor cells have also been studied in tissue culture models. For example, statins, which inhibit 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase, have been shown to extend the life span of endothelial precursor cells and to enhance their mobilization and incorporation at sites of vascular injury in a model of cardiovascular disease.78,79 Studies conducted in cultured endothelial precursor cells have clarified that statins’ effects are mediated by the activation of the PI3-kinase/Akt signaling pathway. Finally, the antibiotic rapamycin has been shown to induce endothelial precursor cells’ apoptosis and to inhibit their ability to differentiate as mature endothelial cells, partly explaining the anti-angiogenic properties of this drug and supporting its use in preventing pathological neovascularization.80

1.5Pericytes

Blood vessels are formed by two cell types: the endothelial cells that line the vascular lumen and the mural cells (pericytes or smooth muscle cells) that wrap the endothelium on the abluminal side of the vessel wall and share the vascular basement membrane. The second phase of the angiogenic process (resolution) involves recruitment and proliferation of mural cells and their attachment to the newly formed capillaries, leading to stable mature vessels with proper directional blood flow.81-83 In retinal capillaries, the mural cells are pericytes. Therefore, substances that stimulate migration or proliferation of pericytes, such as TGF-beta, PDGF, and angiotensin, could also be potentially involved in regulating retinal neovascularization.

Studies in the developing retina have shown that immature vessels that lack pericytes degenerate when exposed to hyperoxia, whereas mature blood vessels with pericytes are resistant to oxygen-induced degeneration.84-87 To explain the mechanism by which pericytes protect the retinal vasculature, Shih et al. showed that the TGF-beta expressing pericytes are specifically attached on vessels that are resistant to oxygen-induced dropout. Their in vitro studies show that TGF-beta induces VEGF and VEGF-receptor 1 (VEGFR-1, flt-1) expression in retinal endothelial cells.88 The VEGFR-1- specific ligand, placental growth factor, acts as a survival factor for endothelial cells in culture.

Understanding the mechanism of pericyte/endothelial-cell interaction is important in the context of oxygen-induced capillary dropout in retinopathy

of prematurity (ROP). A number of in vitro studies have significantly advanced our knowledge of the mechanisms of cell-cell interactions and the factors involved in the ROP pathology. Orlidge and D’Amore used coculture models for a comprehensive in vitro study, which showed inhibition of endothelial cell growth by direct contact with pericytes or smooth muscle cells.89 Co-culture models can also be used to study the role of endothelial cell contact in differentiation of mesenchymal cells to pericytes/smooth muscle cells.90

Pericytes can be isolated by a method similar to that used for preparation of endothelial cells except that the microvessel preparation is directly plated on a gelatin-coated dish without any enzyme treatment.91 Pericytes are identified by their slow growth and robust cytoskeleton structure. Markers include alpha actin and a pericyte-specific ganglioside.92

2.ASPECTS OF ENDOTHELIAL CELL FUNCTION STUDIED IN CULTURE

2.1Permeability

In retina and brain, capillary endothelial cells form the inner blood-retina barrier (BRB) and blood-brain barrier (BBB), respectively, to regulate the exchange of molecules between blood and neural tissues. In both situations, tight junctions prevent the free diffusion of substances from the blood to neural tissues.93,94 It has been suggested that some mechanisms may function differently in the BRB to protect the retina from light-induced oxidative stress.95 Vascular endothelial cell dysfunction and breakdown of the BRB occur in a number of disease conditions including diabetic retinopathy, macular edema, hypertensive retinopathy, branch vein occlusion, and others.93 Vascular leakage contributes to disease progression by inducing edema and tissue damage. At the same time, vascular hyperpermeability is the critical first step in the angiogenic process in that extravasation of plasma proteins provides a milieu that favors neovascularization.96 Activation of plasminogen and matrix metalloproteinases (MMPs) plays a key role in this process by inducing degradation of the ECM and release of growth factors, which stimulates the migration and proliferation of endothelial cells.97

In vitro models have been used to study the regulation of vascular endothelial cell barrier function and to allow experimental manipulations and observations not possible with intact animals. In the Transwell dual chamber

model, monolayers of endothelial cells grown on a porous membrane are situated in a culture dish such that two separate compartments are formed. The upper chamber represents the vascular luminal compartment, and the lower chamber represents the abluminal compartment. Usually, the flux of solutes of different sizes (e.g. sucrose, sodium fluorescein, fluoresceinlabeled albumin or dextran) from the upper to the lower chamber is monitored at timed intervals.98-102 This procedure has been used successfully to study permeability and transport mechanisms of retinal capillary endothelial cells103-105 and to analyze the effects of VEGF, hydrocortisone, or high glucose on the permeability of retinal endothelial cells.106-108 This model has also been used to investigate the functional role of extracellular proteinases such as MMPs and the urokinase/urokinase-receptor (uPA/uPAR) system as mediators of the TGF-beta or VEGF-induced breakdown of the BRB. In vitro studies have shown that astrocytes and

Muller glia express TGF-beta in latent form and that TGF-beta becomes activated when the cells are incubated under hypoxia conditions.42,109 In

separate experiments, retinal endothelial cells were found to express the basement membrane degrading gelatinase MMP-9 when treated with TGFbeta or cocultured with Muller glial cells or astrocytes. Both TGF-beta and MMP-9 increase retinal endothelial cell permeability, and anti-MMP-9 antibody or TGF-beta latency-associated peptide abrogated the TGF-beta effects.45 During retinal disease, glial cell production of active TGF-beta may contribute to breakdown of the blood-retina barrier by stimulating endothelial cell MMP-9 production.

Another prominent extracellular proteinase system that works in concordance with the MMPs is the uPA/uPAR system. This system has also been shown to have a key role in triggering endothelial cell hyperpermeability. Studies of VEGF’s effects on permeability have shown that treatment with VEGF or uPA increases permeability of retinal endothelial cell monolayers, but with different kinetic response patterns.46 The uPA-induced permeability increase is rapid and stable for over six hours. In contrast, the permeability effect of VEGF is biphasic, with an early and transient permeability increase followed by a delayed and sustained permeability increase starting 4-6 hours post VEGF treatment and lasting for 24 hours. Moreover, this delayed phase is accompanied by a decline in transcellular electrical resistance (TER) of the monolayer, which was not seen with the initial permeability increase. It has been shown that the early permeability increase is transcellular and is mediated by cell membranederived caveolae.44 The late phase of the VEGF-induced permeability increase as well as the entire uPA-induced permeability response was shown to involve redistribution of junction proteins and, therefore, is very likely to involve alterations in paracellular permeability through the cell junctions.

Measurement of TER across endothelial cell monolayers has been taken as an indicator of paracellular permeability barrier function and is usually done using hand-held chopstick electrodes. Recently, instrumentation referred to as ECIS (Electrical Cell-Substrate Impedance Sensing, Applied Biophysics, Troy, New York) has been introduced, which can measure both paracellular resistance and the average cell-substrate distance. In ECIS, cells are plated in special 8-well chamber slides equipped with gold plated electrode arrays. The electric current passing through the cell monolayer covering each electrode is measured independently in each chamber. The advantage of the ECIS method over previously used manual methods is that electrical resistance is monitored continuously and in real time, before, during, and after treatments applied to multiple independent chambers. Studies comparing ECIS measurements with those done using the Transwell model have shown that the resistance caused by cell-substrate contact substantially influences the TER data and that the extra resistance due to cell-substrate spaces depends on both cell type and properties of the polycarbonate filter system.110 Studies using the ECIS system should be useful in dissecting the potential contribution of such differences in cellsubstrate attachment to the TER alterations observed in studies of disease models where cell-substrate attachment is likely to be altered (such as diabetic retinopathy).

The ECIS system can also be used for in vitro analysis of cell migration using a “wound-healing” assay in which cell migration is assayed following mechanical disruption. In manual assays, a scrape is made in the cell layer, and the advance of the cells into the wound is assessed by microscopy. Using the ECIS system, the wounding can be accomplished electrically by using high voltage to cause severe electroporation and death of the cells in direct contact with the electrode surface. After this treatment, the migration of the surrounding cells onto the electrode surface can be assayed in real time by charting the recovery of electrical impedance. This procedure has been shown to be highly reproducible and quantitative and to provide data similar to that acquired with traditional measurements.111 Studies in progress using this system with retinal endothelial cells indicate that high glucose increases endothelial cell permeability and migration through induction of uPA/uPAR activation in endothelial cells (Behzadian et al., unpublished).

2.2Extracellular Matrix Proteolysis

As has been explained above, extravasation of blood proteins from leaky vessels provides a favorable environment for the initiation of angiogenesis. In order for angiogenesis to occur, the activated endothelial cells must first detach from the vessel wall and penetrate their basement membranes and the surrounding ECM. The complex composition of the microvascular ECM implies that multiple highly specialized enzymes are required for its degradation. Proteolytic enzymes such as uPA and MMP collagenases and

gelatinases112-115 are produced, bound, and activated by endothelial cells116-118 and mural cells.119,120 These enzymes degrade the basement membrane and

the interstitial stroma of the surrounding tissue in the region of capillary sprouts. Because most of the matrix-degrading proteases are secreted as latent pro-enzymes, the physiological activation, rather than production of the enzyme, is the critical controlling point. Activation of pro-enzymes and zymogens occurs by a cascade of autocatalytic, reciprocal interactions. In the case of pro-MMP-9, this activation depends critically upon binding of uPA with uPAR at the cell membrane (see Figure 2).

Reciprocal Zymogen Activation

Figure 1-2. Reciprocal zymogen activation. Binding of uPA with uPAR initiates the activation of a proteolytic cascade and focuses ECM proteolysis at the plasma membrane.

The MMPs are a family of extracellular proteolytic enzymes that are mainly involved in tissue remodeling. MMP substrates include all forms of collagen and a variety of other ECM components, including ECM bound cytokines and growth factors. MMPs have been found in virtually every tissue of the body under conditions of both health and disease. In retina, MMP activity has been associated with numerous disease conditions, including age-related macular degeneration, proliferative diabetic

retinopathy, glaucomatous optic nerve head damage, vitreoretinopathy, and others (for review see121,122).

The role of the MMPs in angiogenesis has been investigated using an in vitro rat aortic ring model. Inhibition of microvessel outgrowth in this model by MMP inhibitors demonstrated the requirement of MMP activity for angiogenesis.123 These studies also showed that the profile of MMP expression depends both on matrix composition and exogenous growth factors. For example, the gelatinase MMP-2 and the stromelysin MMP-3 were present at high levels during vessel formation in fibrin matrix, whereas the stromelysin MMP-11 and membrane-type-1-MMP were expressed in collagen culture. Basic FGF induced upregulation of gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10 and MMP-11), and the interstitial collagenase MMP-13, whereas VEGF induced expression of MMP-2 only. Such in vitro models provide a basis for developing MMP inhibitors for use as anti-angiogenic therapy.

In vitro studies have indicated that most of the MMPs are induced in the same fashion and by a large number of cytokines or growth factors including IL-1-beta, TNF-alpha, PDGF, EGF, TGF-beta, NGF, and others. However, all MMPs are produced in latent pro-enzyme form. Multiple microenvironmental factors contribute to the temporal and spatial regulation of MMP activation, which is the rate-limiting step in their function. Plasmin and uPA have been implicated in physiological activation of many of the

collagen-degrading MMPs, including MMP-9, which has been associated with pathological angiogenesis in retina.124,125 As has been explained above,

activated endothelial cells express uPAR, which plays a key role in VEGFinduced increases in paracellular permeability.46 Since both plasminogen and pro-MMP-9 bind the cell membrane, uPA binding to uPAR provides a mechanism for the cell to focus a cascade of proteolytic activity at the cell surface. This localization is so precise that it may restrict the enzyme activity to the site of membrane contact with the ECM.

Under normal physiological conditions such as wound healing and tissue remodeling, proteolytic activities are precisely controlled and localized on the cell surface. However, during conditions of pathological

neovascularization, excessive proteolytic activities may contribute to the formation of disorganized, unstable and hyperpermeable vascular tufts, which fail to maintain appropriate levels of tissue oxygenation and nutrient delivery. In other words, excessive proteolysis is incompatible with normal capillary morphogenesis.126 Using a three-dimensional fibrin gel, Montesano and co-workers showed that neutralization of excess proteolytic activity plays a permissive role in angiogenesis and other invasive processes by preventing uncontrolled matrix degradation.127 A number of in vitro models have been devised to investigate production of proteolytic enzymes in vascular endothelial cells and to determine their role in cell migration. Gross et al. found that the capillary endothelial cells produced 5-13 times the basal levels of collagenase activity in response to tumor promoter 12-O- tetradecanoyl phorbol-13-acetate (TPA), whereas aortic endothelial cells and fibroblasts showed a minimal response to TPA.128 Later, using crude angiogenesis stimulating factors, these authors confirmed that induction of plasminogen activator and collagenase activities are limited to capillary endothelial cells.129

2.3Cell Migration

During angiogenesis, proteolysis of the ECM sets the stage for the directional migration of endothelial cells along a concentration gradient of pro-angiogenic factors towards the sites of tissue ischemia. Endothelial cell migration involves temporary attachment and detachment of cell surface adhesion molecules to the ECM. This process is influenced by a number of microenvironmental factors and is associated with changes in adhesion molecules on the cell membrane and rearrangement of cytoskeletal filaments within the cells. In the earliest study to distinguish between the migration and proliferation of endothelial cells during angiogenesis, Schoefl observed that endothelial cell migration was the initiating, and probably the ratelimiting, event in regeneration of capillaries after tissue injury.130 Similarly, Ausprunk and Folkman showed that migrating endothelial cells initiate the extension of capillary sprouts toward the source of the angiogenic factors.131 Ischemia/hypoxia is thought to be a major angiogenic stimulus. The chemotactic factors attracting endothelial cells toward the ischemic tissue have not been fully characterized, but are thought to include VEGF. In vitro models of cell migration allow for the study of the specific angiogenic factors and their interaction with cell adhesion molecules involved in cell migration.

In the wound closure assay of cell migration, endothelial cells are grown to confluence in growth medium and then switched to serum-free medium prior to addition of the factor to be tested. The monolayer is wounded with

an object such as a sterile wood stick or cell scraper. The culture is incubated and periodically monitored under a microscope as the cells move onto the denuded area.132 The effect of fibrin on the migration of bovine aortic endothelial cells was investigated by wounding the confluent monolayer and counting the number of cells crossing the wound border per unit time.133 To assay for migration independent of proliferation, wound-induced proliferation of endothelial cells is inhibited by mitomycin C.

Directed migration (chemotaxis) can be assayed using modified Boyden chambers.134 For these studies, endothelial cells are seeded on porous polycarbonate membranes. Membranes are mounted on plastic rings to form small chambers and to fit the wells of a multi-well tissue culture plate. Test substances are added on the opposite side of the membrane. After a period of stimulation, cells on the attachment side of the membrane are scraped off, and membranes are stained for microscopic analysis. Cells that have migrated through the membrane are counted, and data are expressed as number of cells per high power field. Using this model, Nadal and coworkers showed that angiotensin II, via its AT-I receptor, acts as a chemotactic factor and stimulates migration of retinal microvascular pericytes.135

2.4Cell Proliferation

Following detachment and migration of activated endothelial cells, cell proliferation is the next step in the angiogenic process. There are a number of well-established techniques for evaluating mitotic activity and proliferation of endothelial cells in culture. Some protocols call for synchronizing the cell population by serum starvation or by allowing the cells to grow to confluence in order to render them quiescent before they are treated with mitogens. The cells, arrested in G-0, are then stimulated to enter the S-phase, and they are monitored for DNA synthesis or mitochondrial enzyme activity. Alternatively, counting cells is a direct and simple method in which cells are seeded at low density in normal growth medium and then switched to a serum-free or low serum (0.1-0.5% FBS) medium with or without the regulatory growth factors. Representative sub-confluent cultures

are counted at daily intervals. Cells are removed by trypsinization and counted with a hemocytometer or by using a coulter counter.136,137

Incorporation of thymidine into newly synthesized DNA can also be used to evaluate the mitogenic response of synchronized quiescent cells to various agents. After treatment with mitogen, cells are pulsed by exposure to [methyl-3H]-thymidine for 0.5 to 1 hour, washed thoroughly, and then incubated in normal medium for another 1-3 hours. The monolayer is then