Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

covered and rinsed with a chilled solution of trichloroacetic acid (TCA). The fraction of radiolabeled nucleotide that is incorporated into DNA is TCAinsoluble and remains in the dish. The TCA-insoluble material can be removed by NaOH and quantified by liquid scintillation counting.138 This method has been used to show that human retinal extracts stimulate thymidine uptake in bovine aortic endothelial cells139 and that human growth hormone stimulates thymidine uptake in human retinal microvascular endothelial cells.140 Thymidine incorporation has also been used to show the mitogenic effects of basic FGF and VEGF in retinal endothelial cells and pericytes under normal or hypoxic conditions.141 VEGF and basic FGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced under hypoxic conditions. Moreover, it was found that the proliferation of pericytes was stimulated to a greater extent by basic FGF relative to VEGF. Incorporation of bromo-deoxyuridine (BrdU) is another method for measuring DNA replication in response to mitogenic stimuli. Cells are grown in 96-well microtiter plates to ~50% confluence. The cells are then exposed to particular test agents, and mitotic activity is quantified by using anti-BrdU antibody and a colorimetric substrate reaction.

Another simple method of detecting cell proliferation is to determine cell density in culture using the DNA-enhanced fluorescence assay. Using microtiter plates, this method allows a large number of agents to be tested simultaneously for their effects on cell growth. However, when positive agents are identified by this method, the efficacy of the selected factors must be confirmed by a more direct method such as cell counting. Briefly, fixed cells are labeled with DNA stains such as 4’6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), ethidium bromide, or Hoechst 33342. Then fluorescence intensity is quantified using a fluorometer. This procedure has been used for determining serum-stimulated growth of smooth muscle cells and mitogen-induced growth of endothelial cells.142 The advantage of this method is that the fixed cells can be stored for prolonged periods until all tests are completed, thus allowing time-course proliferation assays with minimal inter-assay variations.

A colorimetric cell proliferation assay, referred to as MTS or MTT assay, is based on the ability of living cells to take up thiazolyl blue and convert it

into dark blue formazan. The reaction is driven by mitochondrial succinate dehydrogenase activity, which can be correlated with cell density.143,144 The

assay has been used to show the mitogenic effects of fibronectin fragments on human retinal endothelial cell proliferation.132,145

2.5Tube Formation

The resolution/differentiation phase of the angiogenic process is characterized by the cessation of endothelial cell proliferation, followed by alignment and differentiation into cords of lumenized vessels. This step of the angiogenic process can be modeled in vitro by monitoring endothelial cell alignment and lumenization in a three-dimensional ECM environment. For this “tube formation” assay, microvascular endothelial cells are grown on a three-dimensional gel consisting of type I collagen,146 Matrigel, or fibrin.147 Cells are grown for 2-5 days in the gels in basal medium and different stimuli are added. Cells invade the matrix and form cords and tubelike structures. Randomly selected fields are photographed by phase-contrast microscopy. The total length of sprouts and numbers of branches per field is measured. Lumenization of the tubes can be verified by microscopic analysis

of tissue sections. The tube formation process can be induced by treatment of cells with tumor promoters, basic FGF,148,149 or other angiogenic factors. In a

study of bovine retinal endothelial cells,150 cells plated within a collagen gel matrix self-associated to form three-dimensional meshworks. This morphogenesis was accomplished by cell migration and did not involve cell proliferation. By contrast, retinal pericytes and smooth muscle cells divided and remained homogeneously distributed when plated within a collagen gel matrix. Moreover, it was found that endothelial migration in collagen gels was induced more effectively by VEGF than by basic FGF and that VEGF and basic FGF have synergistic effects on cell invasion.151 The Matrigel tube formation assay has been used to compare the angiogenic properties of retinal endothelial cells isolated from wild-type and thrombospondin-1- deficient mice. Retinal endothelial cells from wild-type mice formed capillary-like networks on Matrigel, whereas the ability of the retinal endothelial cells from TSP1-/- mice to form capillary-like networks was severely compromised.41 Studies of tube formation in type I collagen have shown that a line of retinal endothelial cells formed capillary-like structures in response to apelin, an endogenous ligand for the orphan G proteincoupled receptor, whereas endothelial cells isolated from human umbilical vein (HUVECs) did not.152

During angiogenesis, the final pattern of blood vessel formation is governed by the concurrent and dual regulation of endothelial cell morphogenesis and regression.131 Endothelial apoptosis has been suggested as a major mediator of vascular regression during normal developmental or pathological vaso-obliteration.153-155 Studies using the tube formation assay have demonstrated that capillary morphogenesis in vitro is associated with apoptosis and that inhibition of TGF-beta signaling inhibits this process.156

A variation of the tube formation assay for analysis of capillary regression was developed by Davis et al.157 The addition of plasminogen to threedimensional collagen matrices was found to result in activation of MMPs, collagen proteolysis, and capillary regression.

2.6Retinal Explants

Organ cultures of intact retinas, partially dissected retinas and retinal slices have been used extensively for research on retinal differentiation, synaptic organization, cell and neurite outgrowth and cell-cell interactions between neurons and glial cells (for review see158). These explant models have the advantage of preserving near normal tissue architecture of the retina in situ. Although these differentiated cultures cannot be propagated in vitro, they can be maintained for periods of days or even weeks. The explant culture models have the disadvantages of greater experimental variability as compared with cultured cells and are difficult to use for some quantitative studies due to variations in tissue geometry and cellular composition. However, the cells in explanted tissues have the advantage of retaining to some extent in vivo histological and biochemical features that are commonly lost when isolated cells are propagated in culture.

The use of retinal explants for studies of retinal angiogenesis has been limited. However, Knott and colleagues have described a human model of retinal angiogenesis159 based on refinement of a bovine retinal explant model developed previously by the same group.160 In this preparation, a 4 mm diameter disc of retinal tissue is placed within a fibrin matrix in medium containing 2.5% platelet-poor plasma and monitored by light microscopy for 1 to 14 days. Immunostaining analysis of tissue sections using antibodies against von Willebrand’s factor, glial fibrillary acidic protein, the macrophage/microglial cell marker (CD68), and the cell proliferation marker Ki-67 demonstrated that vascular growth during the in vitro incubation was correlated with activation of glial and microglial cells. Microscopic analysis of the intact tissue explants revealed obvious growth of vessels from the tissue into the surrounding matrix within 3 days of culture. Immunolocalization of von Willebrand’s factor showed an increase in the number and size of vessels within the inner nuclear layer of the tissue explants. Localized expression of endogenous VEGF was evident after 3 days in culture and was associated with angiogenic growth and glial cell activation as well as with the appearance of immunoreactivity for the monocarboxylate transporter-1 within the retinal endothelium. The expression of this transporter in the retinal endothelium could be attributed to the ability of the endothelium to respond to the demands of glucose metabolism and consequent lactate production in the ischemic retina.159

Although levels of tissue oxygenation were not assessed in this study, it is likely that the increase in VEGF expression and retinal angiogenesis occurred secondary to a condition of relative hypoxia consequent to the thickness and lack of vascular perfusion in the explanted retina. Further study in this model may be useful for clarifying the combined effects of ischemia and fluctuating glucose concentrations on pathological retinal angiogenesis as seen in diabetes. This model may also be useful for comparing the specific patterns of vascular outgrowth and angiogenic sprouting that occur in retina with those that have been observed in other three-dimensional models of angiogenesis, such as the rat aortic ring model (see below). Such studies should help to answer the question of whether or not retinal glia and microglial cells have a specific role in retinal angiogenesis, as has been suggested based on results of previous studies using animal models in vivo and co-culture models in vitro.

2.7Aortic Ring Explants

The rat aortic ring model has proven to be a highly useful method for analysis of the angiogenic process.161-163 In this assay, rat aortic rings embedded in collagen gels have been shown to give rise to a network of branching microvessels composed of a properly polarized monolayer of endothelial cells surrounded by a discontinuous coating of mural cells (pericytes/smooth muscle cells).162 The angiogenic response can be stimulated with angiogenic factors or blocked with angiogenic inhibitors. This model, which was first described in 1982164 and was later modified as a quantitative assay in 1990,161 is now widely accepted as a cost-effective and convenient method of assaying angiogenesis.

While many studies have used the rat aortic ring model to test inducers and inhibitors of angiogenesis,165 its use in studies related to retinal angiogenesis has been limited. In a recent study of the role of VEGF in the guidance of angiogenic sprouting, Gerhardt and colleagues used the rat aortic ring model to show that stalks of vascular sprouts are composed of highly migratory cells and that the tips consist of specialized endothelial cells, which extend numerous filapodia in the direction of their migration.166 Time-lapse imaging revealed protrusion and retraction of lamellipodia from single endothelial cells at the tips of the angiogenic sprouts. The endothelial cells at the sprout tips were negative for mitogenic markers (Ki-67 or phospho-histone staining), whereas conspicuous cell proliferation was seen in the endothelial cells in the vascular stalk behind the advancing tips. This same pattern was evident in the sprouting vessels of the developing retina, indicating that angiogenesis in the aortic ring mimics that in the developing

retina. Studies using this model should be helpful in defining the mechanisms that guide the misdirected cell migration during the angiogenic sprouting process that leads to subretinal and vitreoretinal neovascularization during age-related macular degeneration and diabetic retinopathy.

2.8Molecular Strategies for Studies of Angiogenesis

Molecular approaches to identify and manipulate the expression of genes relevant to angiogenesis have taken good advantage of in vitro models. High-throughput screening technologies such as differential display, serial analysis of gene expression (SAGE), and microarray have helped to determine endothelial-specific genes and the mechanism of their regulation under various diseases conditions.

Functional analysis of genes involves three primary approaches: gain of function, loss of function, and gene silencing. Gain of function studies use gene transfer by viral vectors that allow overexpression of the gene. Conversely, vectors overexpressing a dominant-negative gene can be used for loss of function studies. Delivery of inhibitory RNAs or antisense oligonucleotides can be used to silence gene expression and to determine the role of a specific gene product. In studies of retinal vascular development in mice, Adini and colleagues showed that the deletion of the small GTPase RhoB resulted in retarded retinal vascular development. Inhibition of RhoB in neonatal rat retina using farnesyl transferase induced vascular endothelial apoptosis. To confirm their in vivo data, the authors used both antisense oligonucleotides and dominant-negative RhoB expression to specifically reduce RhoB expression in a primary endothelial cell culture model. These treatments inhibited Akt survival signaling and tube formation and induced apoptosis, confirming a specific role of RhoB in endothelial cell survival.167

Recently, Oshima and co-workers demonstrated that chondromodulin (ChM-I), a cartilage-derived factor that inhibits angiogenesis, is expressed in both cartilage and eye. Others have discovered that tenomodulin (TeM), a protein homologous to ChM-I, is expressed in hypovascular tissues such as tendons and ligaments. To determine if TeM also has anti-angiogenic properties, adenoviral constructs expressing TeM were used to test the effects of TeM in cultured human retinal endothelial cells. It was found that TeM and ChM-I gene transfer inhibits cell proliferation and tube formation in retinal vascular endothelial cells.168

Reich and colleagues performed experiments to show that VEGF siRNA is effective in blocking VEGF expression in a human cell line in which hypoxia was chemically induced by desferrioxamine. The authors showed that VEGF siRNA treatment in vivo blocked hypoxia-induced increases in

VEGF expression in mouse eyes and prevented choroidal neovascularization induced by laser photocoagulation.169

Work in our lab has employed an antisense olignucleotide strategy for blocking gene expression to demonstrate the role of the transcription regulator STAT3 (signal transducer and activator of transcription 3) in the autocrine expression of VEGF in bovine retinal endothelial cells.170 Studies using adenoviral vectors to overexpress a dominant-negative STAT3, which carries a DNA binding mutation, in retinal endothelial cells have demonstrated the specific involvement of STAT3 in high glucose-induced and peroxynitrite-induced VEGF function in these cells.171

2.9Effects of Hypoxia on Vascular Cells

A decrease in tissue oxygen concentration has long been recognized as a primary cause of angiogenesis.172 However, the mechanisms underlying the induction of angiogenesis by hypoxia are still poorly understood. Chronic ischemia is clearly an important factor in induction of angiogenesis. For example, myocardial ischemia is known to result in collateral development and opening of preexisting vessels. Neovascularization also occurs in chronic inflammatory lesions and solid tumors, both of which are associated with tissue hypoxia.173-175 The retinal microcirculation develops late in fetal life and is strongly influenced by oxygen pressure. As the oxygen pressure rises, the progression of vascularization into the periphery of retina is decreased.176 Retinopathy of prematurity is caused by exposure of underdeveloped retinas to high oxygen at birth when the infant is placed in an oxygen incubator. This results in constriction and obliteration of retinal vessels, thus creating retinal hypoxia when infants are returned to ambient oxygen.177 The sudden hypoxic situation introduces an acute insult to undervascularized retina, leading to massive irregular growth of blood vessels, intra-ocular hemorrhage, degeneration of inner limiting membrane, and retinal detachment. In vitro models of hypoxia allow the study of these complicated events at the cellular level.

Cultured vascular cells have been used to explore mechanisms that underlie hypoxia-induced proliferation and to characterize angiogenesisrelated factors released by endothelial, perivascular and glial cells that might play a key role in pathological neovascularization in retina. Lou et al. have shown an increase in DNA synthesis and proliferation of retinal endothelial cells in response to hypoxia exposure (2% oxygen for 4 days).178 A study using retinal endothelial cells maintained in 1% oxygen for 1 hour showed significant increases in the expression of VEGF and VEGF receptors VEGFR-1 and VEGFR-2.179 This study showed that expression of

angiopoietin 1 was low as compared to angiopoietin 2 during normoxia, whereas hypoxia caused increases in angiopoietin 1 and its Tie-2 receptor while angiopoietin 2 was not altered. Work by Nomura and colleagues showed that relative hypoxia stimulated increases in VEGF expression and DNA synthesis in both endothelial cells and pericytes. Antisense oligonucleotides complementary to VEGF mRNAs efficiently inhibited DNA synthesis in endothelial cells cultured under hypoxic conditions, indicating that autocrine expression of VEGF is involved in hypoxia-induced proliferation of endothelial cells.180

Studies using in vitro models indicate that reduced oxygen causes endothelial cell proliferation via upregulation of VEGF. However, the involvement of other growth factors must also be taken into account when considering the processes that occur in vivo. In addition to VEGF, acidic and

basic FGF, EGF, TGF-beta, and PDGF have been implicated in angiogenesis.174,175,181,182 The expression of basic FGF, which may be

involved in all the steps of angiogenesis, has been shown not to be influenced by hypoxia.183 Expression of TGF-beta has also been shown to be unaffected by hypoxia.184 Instead, TGF-beta becomes activated under hypoxia conditions.42 PDGF-B is a major serum mitogen for mesenchymally derived cells. Since PDGF-B is released by platelets as well as by cells involved in inflammatory responses, it has been suggested to play a role in wound healing.185 Although PDGF-B was previously thought to be devoid of mitogenic activity on endothelial cells,186 functional PDGF-B receptors have been shown to be expressed on hyperplastic capillary endothelial cells in malignant glioma, suggesting that autocrine PDGF-B has a role in the proliferation of endothelial cells. Hypoxia-induced up-regulation of PDGF-B has also been reported.183 Available evidence thus suggests that the major autocrine/paracrine growth factors involved in the control of endothelial cell growth under normoxic conditions are basic FGF, VEGF, and PDGF-B. Under hypoxic conditions, induced VEGF and PDGF-B appear mainly responsible for the endothelial proliferation.

Studies on the effect of hypoxia on other retinal cells, such as Muller cells, astrocytes, glial cells, and pericytes, are also important for our understanding of retinal angiogenesis. In vitro studies have shown that

hypoxia stimulates release of angiogenesis-related factors in retinal Muller cells and in pericytes.42,180,187 We have demonstrated the effects of hypoxia

on expression of VEGF and TGF-beta.42 Muller cells isolated from rat retina were incubated under normoxia or hypoxia and the resulting conditioned media were assayed for their effects on growth on BRE cells. Hypoxia was found to activate TGF-beta and to increase VEGF expression by Muller cells. Eichler et al. have shown that, under hypoxic conditions, Muller cells release not only VEGF but also TGF-beta, PEDF, and thrombospondin-1.187

Studies have been done in cultured endothelial cells to explore the potential role of oxidative stress in hypoxia-induced upregulation of VEGF expression and retinal angiogenesis. This work was based on results of in vivo studies in the mouse model of retinopathy of prematurity, which showed that hypoxia-induced increases in VEGF expression and retinal angiogenesis are correlated with increases in superoxide production and upregulation of the NADPH oxidase catalytic subunit gp91phox.188 Moreover, inhibition of NADPH oxidase by apocynin blocked VEGF overexpression and retinal neovascularization. To examine the potential role of retinal endothelial cells in this process, retinal endothelial cells were exposed to hypoxia (1% oxygen, 6 hours), and the effects on expression and activity of NADPH oxidase and VEGF expression were determined. The results showed that hypoxia caused an increase in superoxide generation and VEGF autocrine expression and that both effects were blocked by inhibition of NADPH oxidase with apocynin or a gp91phox blocking peptide (gp91dstat). These observations indicate that NADPH oxidase is a critical source of oxidative stress and a key mediator of VEGF expression during hypoxia.

3.CLOSING

The cultivation of human HeLa cells by George and Margaret Gey of Johns Hopkins University in 1951 was a milestone in the application of in vitro models to the field of biology. Samples of HeLa cells were soon distributed among laboratories throughout the world, and many scientists adopted the culture conditions to grow other human cell lines and to apply tissue culture models in studying virology, pharmacology, toxicology, and genetics. Tissue culture applications have come a long way in the past 55 years, not only by improvement of culture conditions and instrumentation, but also by development of a number of assay systems for monitoring cell behavior under normal and disease conditions.

Large-scale culture of endothelial cells from human umbilical cords by Jaffe and coworkers in 1972 was a turning point for studies of angiogenesis and was soon followed by isolation of capillary endothelial cells from brain and retina. Assays for endothelial cell growth and proliferation, migration, apoptosis, barrier function, tube formation, and interaction with other mural cells have greatly advanced our understanding of retinal angiogenesis under physiological and pathological conditions. At present, we have not only learned how to handle a variety of cells and tissue explants in culture, but we are also more aware of the limitations of the systems, such as the relative advantages and disadvantages of using transformed cell lines vs. primary

cultures for representing in vivo conditions of physiological or pathological angiogenesis. We are aware of the potential artifacts associated with the addition of serum or other crude supplements to the culture, and we are also attentive to practical limitations of gene transfer techniques in certain assay systems.

In parallel, the use of laboratory mice as a model system for studying processes related to human health and disease has also expanded greatly. Naturally occurring mutant mice have been identified and are assisting us in understanding the functional role of particular genes. The advent of transgene mouse technology enables us to selectively manipulate the function of a specific gene and follow its effects on a disease process or to generate a desirable animal model for human disease. Pathologies representing human disease conditions, such as retinopathy of prematurity, age-related macular degeneration, and diabetic retinopathy, can be readily induced in normal and transgenic animals in order to understand gene function in the disease process. Tissue explants and conditionally immortalized cells isolated from such transgenic animals provide valuable in vitro models to complement in vivo studies of the molecular mechanisms of retinal disease.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (NIH-EY04618 and NIH-EY11766), the American Heart Association, and the Juvenile Diabetes Foundation International.

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