Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

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I.GROWTH FACTORS

A. Historical Perspective and Definition of Growth Factor

The study of growth factors originated from attempts to grow dispersed mammalian cells in vitro. It became clear that “factors” present in animal sera were critical for the successful maintenance of cultured mammalian cells. Growth factors have evolved in order to carry out cell-to-cell communication in multicellular organisms. The rate of cell proliferation in unicellular organisms, such as bacteria, is dependent upon and limited by the availability of

nutrients in the environment. However, in multicellular organisms, cells respond to chemical signals primarily synthesized and released from adjacent cells. The cellular response in the “target cell” was historically associated with growth and cell proliferation, hence the designation “growth factor”. However, it is now clear that a whole range of cellular responses can be elicited by these chemical signals such as cell differentiation, transformation, synthesis, secretion, cell death and motility.

Growth factors are defined as extracellular signaling proteins that are involved in cell-to-cell communication. Most growth

factors act on neighboring target cells by binding to specific high affinity plasma membrane receptors. Upon binding to specific receptors, growth factors induce signal transduction pathways leading to activation of effector mechanisms within the responding cell.

The first growth factor to be isolated and studied was nerve growth factor (NGF) (Cohen and Levi-Montalcini, 1957). This was followed shortly by the discovery of epidermal growth factor (EGF) (Cohen, 1965). The observation that clotted sera provided stimulation for mitosis and cell growth while plasma only supported survival led to the eventual discovery of plate- let-derived growth factor (PDGF) (Ross et al., 1974). Since the 1980s over 200 growth factors, cytokines and related proteins have been isolated and studied both in normal tissue and in the diseased state.

This chapter will explore the roles of growth factors in normal ocular function and pathogenesis and the potential therapeutic use of growth factors for the treatment of ocular diseases. We will focus on major growth factors and pathways that are implicated in ocular disease, and therefore are potential therapeutic targets. Due to space limitations, we often cite review articles that include the relevant primary references, and we will not provide an extensive review on the important role of growth factors in ocular development.

1. Growth factor nomenclature

a. General considerations – Even a casual reading of the literature reveals how complex, arbitrary and confusing growth factor nomenclature has become. Part of the confusion stems from the fact that these molecules were isolated and named by several different laboratories and hence received more than one name. Also adding to the confusion is the somewhat arbitrary grouping of growth factors into families and superfamilies based on a variety of inconsistent criteria.

The greatest majority of growth factors have been named based on either (1) the cell type or tissue from which the factor was first isolated (e.g. platelet derived growth factor (PDGF) and brain derived neurotrophic factor (BDNF)); (2) the response elicited in the target cell upon receptor binding (e.g. hepatocyte growth factor (HGF) and fibroblast growth factor (FGF)); or (3) the principal action that is stimulated (e.g. transforming growth factor (TGF) and bone morphogenetic factor (BMP)).

An additional layer of complexity is now appreciated. It is clear that the cellular environment or cellular context in which cells are located can determine the response to a particular growth factor(s). For example, the extracellular matrix (ECM) can play a significant role in how a cell responds to a given growth factor. Thus the “context” in which the cell is located must be taken into consideration. There is a paucity of data in the literature with regards to “cellular context” and growth factor action. We assume, perhaps incorrectly, that cells grown in vitro in a serumless media will respond to a specific growth factor much the same way they would respond in vivo in which both the ECM and other growth factors are acting simultaneously on the target cell.

b. Growth factors and cytokines – One of the most confusing issues with respect to growth factor nomenclature is the use of the term “growth factor” and “cytokine”. In many cases these terms were used interchangeably which led to additional confusion. From a historical perspective, cytokines were defined as extracellular signaling proteins that interacted with cells of the hematopoietic and immune system, while growth factors acted on other cells.

It is now clear that multiple cell types can synthesize, secrete and respond to a variety of growth factors/cytokines. Thus the name given to a growth factor/cytokine should not be restricted to a given function or target cell. However, it is also now clear that the functions of growth factors/cytokines

are so diverse that clear distinctions are no longer possible. For example, it is known that non-immune system cells secrete traditional cytokines (e.g. IL-1 and IL-6) and immune system cells secrete growth factors (e.g. FGF-2 and TGFβ-2). In this chapter the term cytokine will be used to describe a subset of growth factors that primarily regulate hematopoietic and immune system cells.

With respect to cytokines, historically they were called immunocytokines or immunokines. However, it quickly became evident that these chemical messengers could act on cells outside the immune system and the term cytokine became prominent. The term cytokine is a neutral term in the sense that it does not describe the target cell or the response elicited in the target cell. As subdisciplines of science developed, cytokines began to be segregated into specific categories or subdivisions. These subdivisions have evolved in an attempt to define a biologically active factor that acts within a particular setting or disease state. In the field of immunology we refer to interleukins (IL) and colony stimulating factors (CSF) while in virology we described interferons (IF) and in cancer biology we describe tumor necrosis factors (TNF).

Cytokines are a unique family of growth factors. Secreted primarily from leukocytes, cytokines stimulate the humoral and cellular immune responses, as well as the activation of phagocytic cells. Cytokines that are secreted from lymphocytes are termed lymphokines whereas those secreted by monocytes or macrophages are termed monokines. Many of the lymphokines are also known as interleukins (IL) since they are not only secreted by leucocytes, but also are able to affect the cellular responses of leukocytes. Specifically interleukins are growth factors targeted to cells of hematopoietic/immune origin. Over 20 specific IL have now been identified.

c. Neurotrophins and neurotrophic factors –

Another point of confusion is the use of the term neurotrophin and neurotrophic

factor. The term neurotrophin refers to a small family of growth factors that include NGF, BDNF, neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). The term neurotrophin should only be used when describing members of this family. However, it is also clear that other growth factors can have a “trophic” action on neurons or neuroglia. For example, glia derived neurotrophic factor (GDNF) or ciliary neurotrophic factor (CNTF) have clear actions on neurons and are considered to be neurotrophic factors. Thus growth factors that have trophic action on neurons and neuroglia but do not belong to the neurotrophin family are referred to as neurotrophic factors.

B. Growth Factors Signal via Paracrine, Autocrine and Juxtacrine Mechanisms

Growth factors are secreted proteins that usually act over short distances. Historically, the mechanism of action of growth factors was considered to be via paracrine signaling (e.g. cell-to-cell communication via soluble factors). However, it is now appreciated that growth factors can stimulate the same cell from which it has been released via an autocrine mechanism, or can stimulate adjacent cells by direct cell– cell contact via a juxtacrine mechanism without release of the factor. These mechanisms of secretion are summarized in Figure 5.1.

1. Paracrine communication

Paracrine communication describes the action of a secreted growth factor from one cell on a neighboring target cell (Cooper and Hausman, 2004). The definition assumes that the secreted growth factor diffuses over a short distance or is “presented” to the target cell by the ECM and thus they are considered local mediators of cell communication. Growth factors acting via the paracrine mechanism modulate the microenvironment and have been demonstrated to be involved in a variety of processes including angiogenesis, wound

(a)Paracrine cell signaling

(b)Autocrine cell signaling

(c) Intracrine cell signaling

(d) Juxtacrine cell signaling

FIGURE 5.1 Summary of various mechanisms for growth factor signaling between cells. (a) Paracrine signaling involves the secretion of the specific growth factor from one cell and the binding to high affinity receptors of the target cell. This results in a response in the target cell. (b) Autocrine signaling involves the secretion of a specific growth factor from one cell and the binding of the growth factor to high affinity receptors on the same cell. This results in “auto-stimulation” of the cell. (c) Intracrine signaling involves the synthesis of a growth factor and the binding of the growth factor to an intracellular receptor leading to cell stimulation. (d) Juxtacrine signaling involves direct cell-to- cell contact in which the growth factor is membrane bound on cell 1 and the high affinity receptor is in the membrane of cell 2. This interaction results in activation of cell 2

healing, cell motility and metastasis. It was previously assumed that the majority of growth factors act on target cells via a paracrine mechanism. This mechanism of cell-to-cell communication differs from that mediated by endocrine factors that involve the transport of a factor to the site of action by the circulation. For paracrine signaling molecules to be delivered to the proper target cell, the secreted molecules must not be allowed to diffuse too far. For this reason secreted molecules are (1) rapidly taken up

by the neighboring target cell; (2) destroyed by extracellular enzymes; or (3) immobilized by the ECM, often to be released at a later time.

2. Autocrine communication

When the growth factor affects the same cell from which it is synthesized and released, the activity is termed autocrine communication or auto-stimulatory growth control (Cooper and Hausman, 2004). This mechanism of communication can only take place if the cell that releases the growth factor also expresses specific high affinity receptors and responds to the growth factor. This effectively creates an autogenous loop in which a growth factor acts back on the cell that produced it. Both simple and complex autocrine growth control loops have been described. In fact, concentration-dependent complex control of autocrine loops exists. This type of signaling may be part of a negative feedback mechanism. It is also possible that autocrine stimulation leads to uncontrolled cell proliferation, as seen in several cancers.

3. Juxtacrine communication

While most growth factors are secreted from the cell, some growth factors remain associated with the cell membrane rather than being secreted into the ECM. These growth factors are called membraneanchored growth factors. This type of growth factor specifically acts via direct cell- to-cell contact with the target cell and can only signal a cell that contacts the cell. It is also assumed that the target cell expresses specific growth factor membrane receptors. Interestingly, membrane-bound growth factors frequently are incompletely processed biologically active precursors of the secreted form of the growth factor. It is also possible that alternative splicing of the corresponding mRNA generates membranebound growth factors. Membrane-bound growth factors acting via the juxtacrine mechanism appear to elicit the same

spectrum of responses as soluble growth factors. This type of signaling has been demonstrated during development where spatial restrictions are important and in various immune responses.

4. Intracrine signaling

Intracrine signaling is a mechanism of growth control involving the direct action of growth factors within the cell. Some growth factors produce factor/receptor complexes at the cell surface and are rapidly internalized by the cell in question and translocated to the nucleus without degradation. This mechanism differs from autocrine signaling because the growth factor in question is never secreted from the cell. One can think of this signaling pattern as creating an internal autocrine loop requiring the presence of suitable intracellular biologically active receptors. The mechanisms supporting intracrine growth control are largely unknown. However, several growth factors (e.g. CNTF, FGF-1) do not process a secretory signal sequence allowing their release via classical secretory pathways. Intracrine signaling has been suggested as a possible mechanism for the biological activity of these types of growth factors.

C. Growth Factor Families

Growth factors are often grouped as members of larger families/subfamilies of structurally and evolutionarily related proteins. A comprehensive review or listing of all growth factor families is beyond the scope of this chapter. Some of the major families with respect to the pathophysiology and therapeutic targets of the eye will be summarized briefly. These include transforming growth factor β (TGF-β) including bone morphogenetic proteins (BMP), epidermal growth factor (EGF), neurotrophins (NT), fibroblast growth factors (FGF), and vascular endothelial growth factor (VEGF). A summary of members of growth factor families is included as Table 5.1.

TABLE 5.1 Growth factor family members

TGF-β TGF-β1-3, BMP-2 to BMP-20, activins/

1. Transforming growth factor-β (TGF-β)

The human genome encodes at least 42 different members of the TGF-β superfamily of growth factors. The TGF-β superfamily includes TGF-β1-3, activins/inhibins, nodal, myostatin, anti-Müllerian hormone and bone morphogenetic proteins (BMP) (Miyasawa et al., 2002). A distinguishing structural feature of members of the TGF-β superfamily is the presence of seven conserved cysteines. Six of the conserved cysteines are involved in formation of intrachain disulphide bonds that permit folding of the molecule into a unique threedimensional structure called a cystine knot (Cheifetz et al., 1987; Miyazono et al., 2005). The seventh cysteine residue makes a single interchain disulfide bridge between the two subunits. The result is the formation of a covalently linked dimer, which is critical for biological activity (Cheifetz et al., 1990; Gilboa et al., 2000; Miyazono, 2000)

Bone morphogenetic proteins (BMPs) are the largest subfamily of proteins within the TGF-β superfamily and are known to be involved in numerous cellular functions in adult tissues including the eye. Bone morphogenetic proteins were originally identified as osteoinductive growth factors that promoted bone and cartilage formation. However, BMPs are expressed in a number of other tissues, and have been shown to be involved in development, morphogenesis, cell proliferation, apoptosis, and extracellular matrix synthesis. Bone morphogenetic proteins primarily exist as homodimers. The BMPs are synthesized as precursor

proteins that contain a hydrophobic secretory sequence and pro-peptide sequence. The mature BMP protein is located at the carboxyterminal of the precursor molecule and mature proteins are derived from proteolytic cleavage of this carboxyterminal region. Unlike other members of the TGFβ-family of growth factors, BMP proforms do not form latent complexes with their mature counterparts. Cleavage of the variable length pro-segment occurs prior to secretion.

Upon ligand binding, TGF-β/BMP signaling is conveyed from the cell membrane to the nucleus by the Smad family of proteins/transcription factors (Cheifetz et al. 1987, 1990; Gilboa et al., 2000; Miyazono, 2000). An excellent review article on Smad protein/transcription factors has recently been published by Massague et al. and should be consulted for a detailed description of the Smad-dependent signaling pathway (Massague et al., 2007). The Smad protein/transcription factors are subdivided into three separate classes including (a) receptor Smads (R-Smad); (b) common Smad (Co-Smad4); and (c) inhibitory Smads (I-Smad). In Smad-depend- ent BMP signaling, binding of BMP to the constitutively active BMPR-II receptor causes phosphorylation of the GS domain of the BMPR-I receptor. Subsequently, activated BMPR-I receptors dock with and phosphorylate R-Smads. In BMP signaling the R-Smads are R-Smad1, R-Smad5 and R-Smad8 while in TGF-β signaling R-Smad2 and R-Smad3 are utilized (Massague et al., 2007). Phosphorylated R-Smads subsequently assemble with, and form a heteromeric complex with, Co-Smad4. Co-Smad4 is a common partner for all R-Smads. The heteromeric complex consisting of R-Smad1/5/8 and Co-Smad4 protein then translocates into the nucleus to regulate transcription of specific target genes. I-Smad6 and I-Smad7 are inhibitory Smads that block both Smad-receptor and SmadSmad interactions, thus down-regulating BMP signaling.

2. Epidermal growth factor (EGF)

This family of growth factors consists of epidermal growth factor (EGF), transforming growth factor-a (TGF-α), heparinbinding EGF-like growth factor (HB-EGF), amphiregulin (AR), betacellulin (BTC), epiregulin (EPR), epigen and four neuregulins (NRG1-4) (Dreux et al., 2006). Members of the EGF family contain one or more repeats of a conserved six cysteinecontaining motif that is located in the extracellular domain that gives the structure the ability to form three intramolecular disulfide bonds. This organization allows the formation of three loops that is critical for ligand binding to their respective EGF receptor. Specific processing of EGF ligand precursors occurs to give rise to the mature ligand. Members of the EGF family are derived from precursor proteins that are type-I trans-membrane glycoproteins. The precursor molecule consists of three parts:

(a) an extracellular portion containing the EGF ligand; (b) a hydrophobic transmembrane domain; and (c) a cytoplasmic domain (Dreux et al., 2006). The precursor molecules then undergo cleavage that results in the liberation of the mature EGF ligand from the cell membrane. Members of the disintegrin and metalloproteinase family (ADAM) cleave EGF precursor molecules. These molecules are integral membrane proteins that have an extracellular metalloproteinase component that is believed to be involved in the cleavage of the EGF precursor molecule.

The EGF receptor family is composed of four glycoproteins termed ErbB1 (EGFR, HER1), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4). The receptor consists of an extracellular domain of 621 amino acids, a trans-membrane domain of 23 amino acids and a 542 amino acid cytoplasmic domain containing tyrosine kinases activity. A recent review should be consulted for a more in-depth review of EGF receptor structure, ligand-receptor interaction and receptor activation and dimerization (Dreux et al., 2006).

3. Neurotrophins (NT)

Neurotrophins (NTs) constitute a family of polypeptide growth factors that have previously been reported to promote the development, survival, and differentiation of neurons. Four members of this family have been identified in humans: nerve growth factor (NGF); brain-derived neurotrophic factor (BDNF); NT-3; and NT-4.1. NTs bind with high affinity to a specific class of tyrosine kinase (Trk) receptors, as well as to a low affinity receptor, p75 (Barbacid, 1994). Three Trk receptors have been identified, and their interaction with the NTs appears to be specific (Klein et al., 1991). The first Trk receptor discovered was Trk A, the signaling receptor for NGF. BDNF and NT- 4 signal through Trk B, and NT-3 signals through Trk C. In addition to the full-length Trk receptors, truncated isoforms of Trk B and Trk C, each without the Trk domain, have been identified in humans (Shelton et al., 1995). The function of the truncated trk receptors is unknown.

4. Fibroblast growth factors (FGF)

Members of the FGF family of growth factors are expressed in numerous ocular tissues. The function of FGF members in adult tissues include cell proliferation, cell migration, cell differentiation and cell survival. This is a large family of growth factors as currently there are 22 known members in humans that signal through five receptors (FGFR1-5) (Turner et al., 2006). Members of the FGF family share a central domain of 120 amino acids that can bind heparin. This binding helps form stable FGF–FGFR complexes. In addition, each of the five FGFRs is known to have splice variants that adds to the variety and complexity of cellular functions controlled by FGF ligands. The FGFRs are tyrosine kinases, although some of the splice variants lack an intracellar kinase domain and may act as soluble receptors. Activation of FGFRs triggers intracellular signaling through either phospholipase cγ with

subsequent protein kinase C activation or through ras/raf signaling and subsequent activation of the MAPK cascade.

5. Vascular endothelial growth factor (VEGF)

Vascular endothelial growth factor (VEGF) is known to be involved in the pathophysiology of angiogenesis and vasculargenesis including tumorigenesis, vascular permeability and metastasis. The growth factor is a 35–45kDa homo-dimeric glycoprotein that is specific for endothelial cells. It was initially reported to be a vascular permeability factor (VPF) (Senger et al., 1983) but Leung et al. (1989) cloned VEGF-A and reported it also stimulated cell proliferation of endothelial cells. The mammalian VEGF gene yields nine isoforms via alternative splicing including VEGF-121, VEGF-145, VEGF-148, VEGF-162, VEGF-165, VEGF183, VEGF-189 and VEGF-206. One isoform (e.g. VEGF-165b) has been reported to be an inhibitor that binds VEGFR2 but does not activate tyrosine kinase signaling. The VEGF-165 is the predominant form that is involved in angiogenesis associated with tumor progression. Many external stimuli initiate VEGF secretion, including hypoxia, low pH and cellular stress.

Three distinct tyrosine kinase receptors (VEGFR1-3) have been reported for the VEGF ligands (Frumovitz and Sood, 2007). The VEGFR1 receptor has been demonstrated to have both positive and negative effects on angiogenesis. The VEGFR2 receptor is the main regulator of vascular permeability and mitotic action on endothelial cells. The VEGFR3 receptor primarily has its effects on lymphatic vessels. A recent review (Bhistkul, 2007) detailing the biology of VEGF with respect to ocular therapeutics should be consulted for further details concerning VEGF and the eye. In addition, section E.2.c (Retinal and choroidal neovascularization) of this chapter describes in more detail the relationship of VEGF and retinal angiogenesis.