Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

receptor D. A similar inhibition of VEGF by PDGF-BB was seen in a rat model of retinopathy of prematurity, which may be explained by suppression of VEGF upregulation (necessary for retinal NV) following PDGF treatment.68

Using the novel assay of secondary sprouting of retinal endothelial cells on Matrigel, we demonstrated that PDGF-BB was not synergistic with any single growth factor tested, including VEGF, FGF-2, IGF-I, and PlGF.26 However, when PDGF-BB was added to a combination of VEGF, IGF-I, and FGF-2, it significantly and dose-dependently increased secondary sprouting, suggesting that cell exposure to several activating factors is required to produce a full angiogenic response. PDGF may promote angiogenic signaling to a response-sufficient level that was not attained by the combination of other factors, suggesting that it activated certain critical signaling intermediates produced in insufficient amounts by the other factors.

2.3FGFs

Acidic and basic fibroblast growth factors are general mitogens and also promote endothelial cell proliferation. At the same time, unlike VEGF, they may not be able to act as independent angiogenic factors in vivo. Gene transfer and gene expression inhibition experiments have shown that in the retina, overexpression of FGF-2 could not induce NV, and without FGF-2, hypoxia-induced NV proceeded in a usual way.69 Also unlike VEGF,70 FGF- 2 does not appear to be needed for choroidal NV.71 However, cell responses to certain growth factors (VEGF, PlGF, VEGF-C, and PDGF-BB) can be augmented by the addition of FGF-1 or FGF-2 (Table 1). Additionally, FGF- 2 synergy with one of the bone morphogenetic proteins (BMP), BMP-7, was demonstrated in the chorioallantoic membrane assay.72 Interestingly, BMP-7 also enhanced TGF-E1-mediated angiogenesis in this model. Mechanisms of this synergy have been studied only for VEGF and PDGF (see above). Interactions with other factors have been described, but no mechanisms have been uncovered to date.

2.4IGFs

Insulin-like growth factors are potent anti-apoptotic and cell survival regulators. At the same time, IGF-I has received considerable attention as an angiogenic factor, especially in relation to DR.4,41 We have previously discussed how IGF-I can synergize with VEGF in vitro26 and in vivo.41 There is much less information concerning IGF-II. Only recently was it shown that IGF-II effects on vessel growth in mouse skin Matrigel plug assay could be

enhanced by epidermal growth factor (EGF). Interestingly, this phenomenon was reproduced in vivo, but not in vitro.73 The authors attributed the effect of EGF to its ability to decrease expression of IGF-binding protein-3, which is known to suppress IGF activity.

2.5Angiopoietins

Angiopoietins are recently described paracrine angiogenic growth factors that signal through Tie1 and Tie2 receptors and are important for embryonic development of the vascular system.74,75 Ang-1 promotes endothelial sprouting through Tie2 receptor, but at the same time it mediates mural cell recruitment and vessel stabilization. Ang-2, on the other hand, destabilizes

vessels. In concert with VEGF, Ang-2 enhances endothelial cell migration and proliferation, heart angiogenesis, and retinal NV.54,55,74,75 Although there are some data on synergy between VEGF and Ang-1,56 other data55,57 show

that Ang-1 antagonizes VEGF or its combination with Ang-2 (Table 2). Surprisingly, Ang-2 was reported to counteract VEGF+FGF-2-stimulated endothelial cell sprouting in Matrigel,76 possibly by blocking chemotaxis (Table 2). As with other factors, the controversial data may be related to differences in cell types or assays. Recently, it was found in tumors that the ratio of Ang-1:Ang-2 is often shifted toward the prevalence of Ang-2,11 which may be conducive to increased angiogenesis. This agrees well with data on elevated VEGF expression in tumors and synergy between these two factors in the angiogenic process. However, apart from VEGF, the interactions of angiopoietins with other angiogenic growth factors need to be explored in more detail before conclusions can be drawn about their action on the vascular system.

2.6Combined actions of multiple growth factors

Although in most studies only pairs of angiogenic growth factors have been assessed for possible interactions, there are also some data on combinations of more than two factors. Actually, even when only two factors are compared, the general effect of additional growth factors is also being evaluated, since many factors can induce each other’s expression. Such data exist on FGF-2 stimulating HGF expression,77 VEGF stimulating PlGF,44 and HGF stimulating VEGF.46 In our experiments, four to five angiogenic growth factors were added to cultures of retinal endothelial cells. Whereas specific growth factor pairs (VEGF and IGF-1 or PlGF and FGF-2) demonstrated an increased effect on cell migration, proliferation, and secondary sprouting compared to single factors, the combination of four or five factors (VEGF, PDGF-BB, FGF-2, IGF-I, and PlGF) produced a potent

synergistic effect in several assays, including cell migration into the wound, secondary sprouting on Matrigel, and cell proliferation.26 These data fully agree with observations in tumors and in the vitreous of patients with proliferative DR where concentrations of multiple growth factors are elevated simultaneously. They also suggest that more than two growth factors are required for a full angiogenic response. However, mechanisms of synergistic effects of multiple growth factors remain to be elucidated.

2.7Extracellular matrix and growth factors

It has long been recognized that extracellular matrix (ECM) and basement membrane proteins play an important role in cellular behavior. In recent years, several ECM proteins have been shown to possess angiogenic (laminin, tenascin-C) or anti-angiogenic (thrombospondins, chondromodulin- 1, secreted protein, rich in cysteine (SPARC, also known as osteonectin), fragments of perlecan (endorepellin), fibronectin (anastellin), type IV collagen (tumstatin, canstatin, arresten), and type XVIII collagen (endostatin)) properties.34,78-85 Many effects of ECM proteins are mediated

by their binding to integrin receptors that transduce signals inside the cells.34,80 At the same time, many aspects of ECM protein involvement in

angiogenesis remain obscure.

In the context of this review, it would be logical to analyze the data pertaining to the interactions of ECM proteins and growth factors. We have shown that tenascin-C, together with VEGF, increases the complexity of tubular structures (number of branching points) formed by retinal endothelial cells on Matrigel.78 The effect on tube network formation was additive. A subsequent study by another group using tenascin-C knockout mice suggested that this ECM protein was involved in the regulation of VEGF. Lack of tenascin-C reduced both angiogenesis and VEGF expression.79

Laminin-1 was shown to synergize with FGF-2 in promoting chick chorioallantoic membrane angiogenesis.81 Moreover, laminin-1 was able to increase FGF-2 and FGFR1 expression during tube formation of endothelial cells in collagen gel. Interestingly, in these systems, laminin-1 did not regulate FGF-2 signal transduction.

Most recently, VEGFR1, which can inhibit the angiogenic activity of VEGF-A, was shown to interact with SPARC. As a result of this interaction, VEGFR1 activation was silenced and VEGF-mediated choroidal NV was promoted.85 Therefore, SPARC can synergize with VEGF-A in inducing choroidal NV by interacting with a VEGF receptor.

Concerning the anti-angiogenic ECM proteins and protein fragments, the data on their interaction with growth factors are scarce. It was shown that canstatin and endostatin could inhibit the expression of many key protein

kinases that are activated by angiogenic growth factors. Endostatin is also known to downregulate VEGFR2.80 Theoretically, since many growth factors utilize the same or similar signaling pathways, these anti-angiogenic ECM proteins might inhibit the action of several growth factors simultaneously.

3.MECHANISMS OF GROWTH FACTOR SYNERGY AND STRATEGIES TO IDENTIFY SYNERGISTIC INTERACTIONS

Most angiogenic growth factors described above have cell surface receptors, the majority of which are protein kinases. These receptors transduce signals inside the cells. Therefore, it is logical to look for mechanisms of growth factor synergy in the signaling pathways and their regulation. Considerable progress has been made in dissecting growth factor signaling pathways, and many of these are reviewed in other chapters of this book. At the same time, the situation is complicated by the fact that many angiogenic mediators have more than one receptor, and these receptors may signal differently. In the case of VEGF, members of the Src family, such as Fyn and Yes, are phosphorylated in response to VEGF-VEGFR1 but not to VEGF-VEGFR2 interactions. In contrast, phosphorylated VEGFR2 associates with Shc, Grb2, and Nck, but also with the phosphatases SHP-1 and SHP-2.86 The available evidence suggests that depending on the situation, VEGFR1 may be either a positive or a negative regulator of angiogenesis, unlike VEGFR2, which seems to be always pro-angiogenic.31

Moreover, different growth factors may use the same pathways for migratory, mitogenic, or anti-apoptotic effects.87 However, depending on the growth conditions, cell type, and receptor involved, the actual pathway used, and hence the outcome, may vary greatly. In muscle cells, for example, PDGF activates the ERK mitogenic pathway in a sustained way, but only transiently activates the Akt survival pathway. In the same cells, IGF-I mainly activates the Akt pathway, consistent with its anti-apoptotic role, and only transiently stimulates ERK signaling.88 Such peculiarities of signaling by different growth factors and their receptors may form the basis of growth factor synergy, whereby growth factor combinations are more potent in their action on cells than single factors.

Another recently discovered oddity is the existence of anti-angiogenic isoforms of angiogenic growth factors, such as VEGF165b, which can bind to but cannot activate VEGF receptors.89 There is also evidence for angiogenic activity of known anti-angiogenic factors, such as PEDF.58 These findings may explain a vast diversity in angiogenic effects from the same

factor and underscore the difficulties in understanding the fine-tuning of normal and pathological angiogenesis that involves the concerted action of various growth factor isoforms.

For therapeutic purposes, it is important to be able to predict whether particular growth factors will enhance or inhibit each other’s effects in specific angiogenic situations. It is logical to assume that if two angiogenic factors have significantly different intracellular signaling pathways, they may synergize. Otherwise, factors that have many common intracellular targets may exert additive effects at best. Because of the complicated nature of signaling pathways, however, it would be difficult, to check signaling intermediates one by one. To circumvent this problem, gene microarray technology is being used to predict complex outcomes by assessing gene expression levels of thousands of genes after administration of single or combined growth factors or anti-angiogenic drugs.

This approach has been successfully used by Gerritsen’s group for VEGF and HGF in umbilical vein endothelial cells.47 It was shown that there was little overlap between genes upor downregulated by each growth factor. When two factors were combined, however, there was a dramatic increase in the number of altered genes, including those related to the cell cycle. These

data fully agree with other findings showing biological synergy of VEGF and HGF.46,48,49 A subsequent gene array study using VEGF and PlGF also

showed that they regulated non-overlapping gene sets in endothelial cells,90 a finding that explains their synergy.37,44

Microarrays were also used to test whether anti-angiogenic compounds would synergize in their action when used in combination.91 It was shown that two anti-angiogenic drugs, thrombospondin-mimetic peptide (DI-TSPa) and TNP-470, changed the expression of very similar sets of genes in cultured microvascular endothelial cells. At the same time, endostatin induced changes in a different set of genes. When tested in combination in mice bearing Lewis lung carcinoma, DI-TSPa and TNP-470 modestly inhibited tumor growth and angiogenesis. However, a combination of either drug with endostatin resulted in a significant inhibition of tumor growth and angiogenesis.

Gene microarray studies have thus shown the power of this methodology for identifying the molecular mechanisms of growth factor synergy. Hopefully, this approach will be expanded to examine other growth factor combinations. With the emergence of custom arrays, including those with genes involved in cell signaling, and of proteomic arrays, we predict a rapid development of our knowledge of molecular mechanisms of growth factor interactions, both synergistic and antagonistic.

4.PROSPECTUS

It is now well established that normal and pathological angiogenesis is controlled in large part by various angiogenic growth factors. There is a growing body of evidence that at sites of pathological NV, concentrations of various growth factors are significantly increased. Moreover, there are many documented cases of concerted action of different growth factors/cytokines in angiogenesis and NV both in vivo and in vitro. Gene array-based strategies for identifying potential growth factor synergies have been developed. For therapeutic purposes, there is a need to enhance angiogenesis (e.g., after myocardial infarction) or, in other situations, to inhibit it (tumors, proliferative retinopathies). New efficient approaches to regulate the angiogenic process must take into account the fact that angiogenic growth factors act in concert during angiogenesis. So far, attempts to inhibit unwanted angiogenesis in the eye and tumors had only partial success, and most clinical trials showed modest effects. This may result from inhibiting an incomplete set of growth factors. Possible future approaches to fight or stimulate angiogenesis can be divided into two major groups. Below, inhibition of angiogenesis will be discussed because this is the most clinically relevant approach in ocular diseases.

First, efforts may be concentrated on local inhibition of master regulators that activate a variety of angiogenic growth factors and cytokines. One such regulator is transcription factor HIF-1D, which is induced by hypoxia. It is well known that many retinopathies (and tumors as well) develop on a

hypoxic/ischemic background. HIF-1D is capable of inducing expression of a variety of growth factors, especially VEGF.35,92,93 An adenovirus-mediated

gene transfer of constitutively active HIF-1D can induce growth factor expression and angiogenesis even in a nonischemic tissue.93 It can also improve perfusion and arterial remodeling in ischemic limbs.94 The data are

emerging that show prevention of growth factor activation in hypoxic conditions and inhibition of unwanted NV by blocking HIF-1D.35,95 This

transcription factor appears to be a very promising target for local stimulation or inhibition of NV for therapeutic purposes.

Another molecule that regulates phosphorylation and activity of various growth factors and their downstream signaling is a ubiquitous protein kinase CK2, formerly casein kinase 2. It has more than 300 substrates in the cell and participates in cell migration, proliferation, differentiation, and apoptosis. CK2 phosphorylates many important signaling intermediates of

angiogenic growth factors, including PKC, Akt, Raf, S6 kinase, p38 MAPK, and insulin receptor substrate-1 (see Table 2). 96 Specific CK2 inhibitors can

significantly reduce ischemic retinal NV in vivo, as well as cell migration and proliferation in vitro in response to several growth factors (Table 2).96

Recent exciting data show that CK2 can regulate the phosphorylation and activity of HIF-1D thereby potentially influencing a variety of angiogenic growth factors.97 Therefore, CK2 may be another promising candidate for inhibition in order to fight unwanted angiogenesis.

Somatostatins represent yet another class of potent anti-angiogenic molecules. They can prevent growth hormone action, which inhibits angiogenesis by some still obscure mechanisms. One example of such drugs is octreotide (Novartis). It can inhibit endothelial cell proliferation stimulated by various growth factors (Table 2).98 This property may explain beneficial effects of octreotide on patients with proliferative DR.99

Targeting one factor to inhibit or enhance angiogenesis may not be enough due to growth factor interactions. With this in mind, combination therapy should be seriously considered. This principle is now routine in treating cancer and AIDS and should be more widely used to fight ocular NV. Our data show that octreotide combined with a CK2 inhibitor, emodin or tetrabromobenzotriazole (TBB), was more potent in inhibiting mouse ischemic retinal NV than either compound alone. Moreover, a low dose of octreotide combined with either CK2 inhibitor could achieve the same extent of NV inhibition as a 5-fold higher dose of octreotide alone.100 Most recently, it was also shown that combined inhibition of VEGF and PDGF-B blocked experimental ocular NV more potently than inhibition of each growth factor alone. These data confirmed the validity of a combination therapy approach against NV.101

Anti-angiogenic combination therapy may thus be considered as a promising approach for fighting retinal neovascular disorders. Because tissues and tumors can acquire resistance to anti-angiogenic drugs,102 combination therapy may soon become the strategy of choice in the therapeutic regulation of angiogenesis.

ACKNOWLEDGMENTS

The author’s research was supported by NIH EY12605, NIH EY13431, Cedars-Sinai Department of Surgery seed grants, and the Skirball program for Molecular Ophthalmology.

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