- Mechanisms of Growth Factor Action
- Growth Factors, Atherosclerosis, and Restenosis After Angioplasty
- Growth Factors, Myocardial Ischemia, and Angiogenesis
- Growth Factor Antagonism
- Growth Factor Agonism
- Growth Factor Therapy: Strategies and Current Limitations
- Selected Abbreviations and Acronyms
- Figures & Tables
- Info & Metrics
Abstract Peptide growth factors are involved in fundamental cellular processes relevant for cardiovascular physiology and pathology, namely, atherogenesis and angiogenesis. The modulation of growth factor–related signals represents a novel strategy for the treatment of cardiac and vascular disease. Experimental modulation of growth factor action has already provided a better understanding of cardiovascular biology and pathophysiology. In turn, the development of specific and powerful molecular tools is setting the stage for the exploration of their clinical potentials. Current strategies include the use of recombinant proteins, specific inhibitors of protein-protein interactions, tyrosine kinase inhibitors, the generation and application of dominant-negative molecules, the development of antisense strategies, and a variety of different gene transfer approaches. Parallel avenues of research are heading toward the same goal, the specific suppression of potent pathogenic stimuli that induce and promote atherogenesis or the augmentation of beneficial ones such as induction of therapeutic angiogenesis. The successful application of one of these strategies seems to be in reach and will certainly be a milestone in molecular medicine.
One decade ago, the first clinical trials were conducted to test the potential clinical benefits of recombinant growth factors. Such studies were initially carried out using hematopoetic growth factors such as granulocyte colony–stimulating factor.1 These studies have proven the usefulness of growth factor application for certain hematological disorders. The functional role of some growth factors in various types of cancer has been recognized,2 and strategies to interrupt either growth factor–dependent autocrine tumor growth3,4 or growth factor–dependent tumor angiogenesis5 are being explored. The involvement of growth factors in the pathogenesis of cardiovascular diseases is increasingly recognized,6 including the propagation of diseases such as atherogenesis and restenosis. In addition, growth factors are involved in the stimulation of functional repair processes, including the formation of a functional collateral circulation in the chronic ischemic myocardium.7,8
As are highlighted in this review, the four different growth factors (PDGF, bFGF, VEGF, and TGF-β) and their different isoforms, receptors, and signal transduction machineries play important roles in the cardiovascular system, namely in atherogenesis and angiogenesis. All are involved in the functional regulation of one or more important structures of the vessel wall, namely VSMCs, endothelial cells or the extracellular matrix (Table 1⇓). The purpose of this review is to discuss current options and potential strategies for molecular interventions related to growth factor action, setting the stage for the development of novel therapeutic strategies.
Mechanisms of Growth Factor Action
Growth factors are potent regulators of cellular function, including proliferation, migration, differentiation, and survival/apoptosis. Growth factor stimulation of cells is a complex and multistep action that transmits extracellular stimuli into the target cell by using various signaling cascades. To better understand the mechanisms and components that form the basis of any molecular strategy of interference, the principals of these cascades are introduced in brief (Fig 1⇓). Binding of growth factors to their specific, membrane-bound cell surface receptors is a key event in the activation process. Binding of growth factors as PDGF, VEGF, or bFGF to receptors of the transmembrane tyrosine kinase type initiates a dimerization process, which results in the activation of the tyrosine kinase domains of the receptors.9 As a consequence, specific signal transduction cascades become activated. They can reach the nucleus, where proliferation and differentiation are modulated, or they can directly affect the function of cellular proteins such as enzymes (eg, PI3K) or cytoskeletal proteins. The modulation of gene transcription can even give rise to indirect effects of growth factors because the expression of growth factors and their receptors can be regulated by other growth factors (Table 2⇓).
The majority of growth factors, such as PDGF, bFGF, or VEGF, stimulate proliferation and migration and inhibit apoptosis. They are positive regulators of the cell cycle. The direct association between growth factor signal and cell cycle regulation, however, is only partially understood. Growth factor stimulation finally results in the production and activation (ie, phosphorylation) of early nuclear proteins that in turn induce transcription of the genes for cyclins, CDKs, and other cell cycle regulators.10 Within the cell cycle, there are two brake-points (G1/S, G2/M) through which the cell must pass before it can enter cell division. Progress through the cell cycle requires the presence of active CDKs, which are activated by cyclins themselves. In response to growth factor stimulation, active CDKs inactivate (ie, phosphorylate) retinoblastoma protein (pRB), a central regulator of the cell cycle. In the quiescent cell, active pRB inhibits cell division by directly inactivating the specific growth-promoting protein E2F, a potent transcription factor. Likewise, overexpression of active pRB inhibits growth factor–stimulated cell division.11 As the central stimulators of the cell cycle, CDKs are functionally regulated themselves by CDK inhibitors such as p21 and p27Kip1. Although overexpression of p21 inhibits cell cycle progression,12 lack of p21 finally results in programmed cell death/apoptosis.13 Similarly, disruption of p27Kip1 in mice gives rise to increased cell proliferation, multiple-organ hyperplasia, and increased body size.14
Unlike PDGF, bFGF, and VEGF, members of the TGF-β family are negative regulators of the cell cycle that lead to growth arrest by directly affecting the cell cycle (eg, activation of CDK inhibitors), but they have a number of other functions as well. There are three different gene products (TGF-β1, TGF-β2, TGF-β3), which usually form homodimers but can also form heterodimers. TGF-β binding induces the formation of hetero-oligomeric complexes of different type I and type II serine/threonine kinase receptors,15 which can signal via Smad proteins, a new class of transcription factors.16 Most of the effects of TGF-β in the cardiovascular system known to date have been gathered for TGF-β1.
The model of interaction of growth factors with their receptors is complicated by the fact that there may be different isoforms or splice variants that can bind to the same or different receptors. This is of functional importance if a therapeutic strategy is aimed at the blockade of a specific growth factor/growth factor receptor interaction. In the case of PDGF, for example, two different genes encode for two different protein subunits (PDGF A-chain, PDGF B-chain), which can form either homodimers (PDGF-AA, PDGF-BB) or heterodimers (PDGF-AB).17 In addition, there are two different receptor genes giving rise to two different receptor subunits (PDGF α-receptor, PDGF β-receptor). As a consequence of different affinities between various ligand and receptor subunits, PDGF-BB can bind to all PDGF receptor subunits, whereas PDGF-AA can bind only to PDGF α-receptor homodimers, and PDGF-AB cannot bind to PDGF β-receptor homodimers.
Growth Factors, Atherosclerosis, and Restenosis After Angioplasty
There is a large body of data available that link growth factor activity to human atherosclerosis and restenosis after angioplasty (reviewed by Ross6). PDGF,17,18 a strong mitogen for mesenchymal cells such as VSMCs and fibroblasts, is regarded as an important mediator of proliferative activity in atherogenesis and restenosis.6 The PDGF-BB isoform is the most potent chemotactic agent known for VSMCs.19 Significant amounts of PDGF are present in the α-granules of platelets, but a number of other cell types, such as VSMCs,20 leukocytes, and endothelial cells, do produce and secrete PDGF.18 Increased levels of PDGF mRNA, PDGF protein, and PDGF β-receptor protein can be detected in the atherosclerotic plaque. On the other hand, the endothelium of large vessels does not express PDGF β-receptors, and PDGF α-receptors could be detected only in injured endothelium.21 In the animal model, the application of PDGF protein22 or enhancement of PDGF expression in the vessel wall23 was sufficient to cause neointima formation. On the other hand, inhibition of PDGF activity or inhibition of PDGF β-receptor production in vivo resulted in the suppression of arterial remodeling and intimal thickening24,25 (Table 3⇓). It has been claimed that the low-molecular-weight compound trapidil (triazolopyrimidine) prevents restenosis after percutaneous transluminal coronary angioplasty.26 Trapidil was initially described as a PDGF antagonist,27 but the molecular basis of its action remains to be established. Similar results have been obtained for bFGF, which is lacking a signal sequence and is found in the basement membranes and could be released as a result of cell injury. bFGF was found to stimulate neointima formation28 (Table 3⇓) and to prevent apoptosis in VSMCs.29 In turn, inhibition of bFGF in these cells induces apoptosis, or programmed cell death.29 Besides proliferation, apoptosis is being regarded as an important mechanism that regulates intimal thickening by modulating the cellularity of the lesion.30
Like PDGF, molecules of the TGF-β family31 are involved in promoting the pathogenesis of atherosclerosis.6 TGF-β exerts three different actions relevant for cardiovascular pathology. It stimulates the formation and deposition of extracellular matrix.32 TGF-β modifies the response of the immune system and suppresses inflammatory processes,33,34 which is thought to have strong implications for the atherogenic process, as suggested by the finding of elevated C-reactive protein in unstable angina.35 In addition, TGF-β can suppress the proliferation of VSMCs. It was suggested that VSMC proliferation inversely relates to the serum levels of lipoprotein(a), which inhibits plasmin formation and therefore the activation of TGF-β.36 Also, TGF-β has been implicated in playing the potential role of repairing ischemic injury because it acts in a cardioprotective manner by reducing the amount of superoxide anions.31 Targeted expression of TGF-β1 promotes vascular endothelial cell DNA synthesis,37 and TGF-β1 gene transfer to the arterial wall stimulates neointima formation in vivo,38 as does the prolonged administration of TGF-β1 protein39 (Table 3⇑). Taken together, the inhibitory effects of TGF-β1,40 PDGF, and possibly bFGF represent promising approaches to slowing down the atherogenic process or preventing restenosis.
Growth Factors, Myocardial Ischemia, and Angiogenesis
Atherosclerosis in the heart leads to progressive narrowing or occlusion of coronary arteries, finally resulting in regional myocardial ischemia. Nature has created mechanisms to partially adapt to regional ischemia through “compensatory angiogenesis” (ie, development of a functional collateral circulation), which is driven by angiogenic factors, most of which represent members of the peptide growth factor family.8 However, this adaptive process is rather slow and unable to compensate fully for the effects of ischemia induced by acute occlusion of a coronary artery. It therefore seems to be logical to substitute the angiogenic factor where it is actually needed and where other means of revascularization, such as angioplasty or coronary artery bypass grafting, are not applicable. This novel concept of the therapeutic induction of neovascularization, also denoted “therapeutic angiogenesis” or “molecular bypass grafting,” has been demonstrated in a number of different animal models to rapidly induce collateralization in the ischemic area (ie, revascularization) and thereby improve organ function (Table 4⇓).
One angiogenesis-inducing factor is VEGF (reviewed by Thomas41), also identified as vascular permeability factor and vasculotropin. VEGF is an endothelial-specific growth factor that has been shown to be involved in both adaptive and tumor angiogenesis. VEGF exists as five isoforms produced by alternative splicing of mRNA encoded by the same gene.41A Recently, two distinct genes denominated VEGF-B42 and VEGF-C43 have been described; however, their roles in angiogenesis and endothelial regulation remain to be determined. VEGF (or VEGF-A) is upregulated under hypoxic and ischemic conditions in vitro44 and in vivo,45 on both the transcriptional and post-transcriptional level.46 Interestingly, not only the ligand VEGF but also its major signaling receptor KDR/Flk147 is upregulated under hypoxia, which has been shown on the transcriptional level in vivo48 as well as on the protein and the functional level in vitro.49 The exact function of Flt-1 is currently unknown, but it does induce tissue factor expression in endothelial cells as well as in monocytes, and it is involved in monocyte chemotaxis.50 The application of VEGF protein to ischemic limbs51 or ischemic myocardium52–54 in different animal models has proven to be sufficient for the induction of neovascularization, resulting in enhanced tissue perfusion and improved function (recently reviewed by Engler55). Besides its activity in promoting neovascularization, VEGF has been shown to stimulate regrowth of the endothelium after angioplasty,56,57 which is believed to protect the vessel wall against remodeling. Besides KDR and Flt-1, two other receptor tyrosine kinases, Tie-1 and Tie-2, show an endothelial cell–specific expression pattern. Although they play a crucial role in the development of the cardiovascular system (embryonic angiogenesis),58,58a their role in compensatory angiogenesis is currently unknown, and so is the role of the recently identified Tie-2 ligand angiopoietin-1.59
bFGF acts directly on both smooth muscle cells and endothelial cells; its action in the vessel wall is therefore not cell type specific.60 bFGF is a potent stimulator of angiogenesis61 and had been applied to induce therapeutic angiogenesis in various animal models62–71 (Table 4⇑). Although some studies convincingly showed the true formation of new capillaries (ie, capillary number per fiber number), others62 did not. Differences in the study protocols (dosage; mode, and time point of growth factor application) might explain these differences. In addition to the angiogenic effect, a direct cardioprotective effect of bFGF has been suggested. With a canine coronary occlusion/reperfusion model, intracoronary application of bFGF resulted in a reduction of infarct size after 7 days, whereas angiogenesis was not (yet) detectable.72 Recently, another member of the FGF-family, namely FGF-5, has been used to stimulate therapeutic angiogenesis through adenovirus-mediated gene transfer in a porcine model of myocardial ischemia.73 Although indirect parameters such as left ventricular function (as determined by echocardiography) indicate a positive effect of the FGF-5 treatment, classic parameters of induced angiogenesis, such as capillary number per fiber cross-sectional area or capillary number per fiber number, remained unchanged. In vitro, the combination of VEGF and bFGF represents the most potent angiogenic stimulus currently known.74 In addition, bFGF was found to upregulate the expression of VEGF75 (Table 2⇑). However, bFGF has been shown to promote atherogenesis and intimal hyperplasia,28,60,76 which is potentially limiting in its use for the stimulation of therapeutic angiogenesis compared with VEGF. Currently, this issue is unresolved.77 The same is true for PDGF, especially for the PDGF-BB isoform,22,23 which has been shown to be a stimulator of collateral formation78 and formation of functional vascular anastomoses.79 Microvascular endothelial cells express PDGF β-receptors80 mediating the angiogenic stimulus of PDGF.81 Moreover, PDGF-BB is able to upregulate bFGF,82 FGF receptor-1,83 and VEGF.82,84 Therefore, PDGF may act as an indirect inducer of angiogenesis as well.
The formation of functional collaterals in the myocardium or ischemic limb, however, is not the only aspect in which angiogenesis takes place in the cardiovascular system. The formation of vasa vasorum within the vessel wall is increasingly recognized as a functional and morphological aspect of advanced atherosclerosis that may trigger complications such as rupture of the plaque, consecutive thrombosis, and tissue infarction.85 The mediators of this process are currently unknown, but VEGF is potentially involved in this process while being produced by arterial smooth muscle cells.86 Likewise, FGF-1, another member of the FGF family, contributes to the formation of vasa vasorum because overexpression in the vessel wall leads to capillary formation.87 In this model, stimulation of plaque angiogenesis was associated with enhanced neointima formation, raising the possibility that enhanced perfusion of the plaque results in an accelerated atherogenic process. The task of functional interference with plaque angiogenesis has not been addressed yet. It is an interesting perspective, that the inhibition of plaque angiogenesis represents a potential means of reducing the progression of atherosclerosis and limiting its complications (Fig 2⇓). On the other hand, promotion of plaque angiogenesis and neointima formation might be unwanted side effects of therapeutic angiogenesis.
Growth Factor Antagonism
A large variety of different approaches has been developed to antagonize the action of growth factors, as listed in Table 5⇓ and illustrated in Fig 1⇑. Examples are given preferentially for PDGF and VEGF.
Functional inhibition of growth factor binding to its receptor or receptors is a potent strategy that can be realized by a variety of different compounds, some of which are purely experimental and some of which could be further developed and explored with regard to their clinical potentials. Neutralizing antibodies recognizing either PDGF or VEGF have been used to antagonize the function of these growth factors in vitro and in vivo. PDGF antagonism results in the inhibition of neointimal smooth muscle cell accumulation secondary to angioplasty and consecutive suppression of arterial remodeling in the rat,24 and inhibition of VEGF leads to reduced vascular permeability.88 Binding inhibitors such as polyanionic substances like neomycin89 or synthetic peptides derived from a growth factor sequence that specifically block the interaction of PDGF with its receptors90 can revert the action of PDGF in vitro. Neomycin was even found to discriminate between the two subtypes of PDGF receptors because it prevents binding of PDGF-BB to the PDGF β-receptor but not to the PDGF α-receptor.89 Heparin-mimicking compounds have been shown to inhibit the interaction of heparin-binding growth factors to the extracellular matrix (also referred to as “low-affinity receptors”), exerting an antiproliferative activity to VSMCs.91 Suramin, a polysulfonyl-naphthyl-urea compound originally developed as an anti-trypanosomal agent, is a potent compound interfering with the binding of a variety of growth factors to their high-affinity receptors and consecutively inhibiting growth factor action. This has been shown for PDGF,92 bFGF,93 and VEGF in vitro94 as well as in vivo.95 In an endothelial denudation model in the rabbit, suramin was shown to inhibit intimal thickening.96 In a similar way, 2-bromomethyl-5-chlorobenzene sulfonylphthalimide antagonizes PDGF action in vitro in a rather specific fashion, inhibiting intimal lesion formation in vivo.97 Finally, a novel group of functional binding inhibitors are represented by high-affinity RNA or DNA ligands, which have the potency of functionally antagonizing VEGF98 and the PDGF B-chain.99
Growth factor receptor blockers of the tyrphostin class100 or the 2-phenylaminopyrimidine class101 of PTK inhibitors have turned out to be promising candidates for a clinically useful strategy. These low-molecular-weight compounds inhibit the enzymatic activity of tyrosine kinases, which, as shown for PDGF, work in the in vitro situation.3,102 Some, but not all, however, work in the in vivo situation as well.103 Recently, it has been possible to identify compounds (eg, AG1296) with high selectivity even between closely related protein tyrosine kinases such as the PDGF receptors and the VEGF receptor KDR.3 This finding provides the basis for specific, differential approaches such as inhibition of restenosis, which is based on the postulate that PDGF-dependent proliferation and migration of smooth muscle cells are inhibited, whereas VEGF-dependent regeneration of the endothelium should be unaltered in large vessels (Table 1⇑).
Mutations or truncations within the growth factor or growth factor receptor molecules can abolish their function. The PDGF β-receptor,104 the VEGF receptor Flk-1/KDR,5 but also the TGF-β type II receptor105 made devoid of a functional kinase domain (ie, dominant-negative growth factor receptor mutants) make cells unresponsive for the corresponding ligand. When overexpressed in the target cell, growth factor binding results in the formation of nonfunctional heterodimers with the wild-type receptor, unable to induce receptor autophosphorylation and activation. A dominant-negative Flk-1 mutant has been shown to inhibit glioblastoma growth through inhibition of angiogenesis in vivo.5 A conceptually different approach is the use of soluble growth factor receptors, which bind the ligand in the liquid phase and therefore competitively prevent ligand binding to functional cell surface receptors, as shown for PDGF.106 Recently, several endogenous soluble Flt-1 molecules have been identified.107,108 Because of their high affinity to VEGF, they are ideal candidates for VEGF inhibition. Their function and in vivo regulation are not known yet; however, in vitro studies using soluble Flt-1 receptors demonstrate their potency as functional antagonists of VEGF-induced proliferation107 and migration of endothelial cells (unpublished results). The concept of dominant-negative mutant molecules has also been explored on the ligand side. PDGF-0, a mutant PDGF molecule that is unable to dimerize and induce dimerization of PDGF receptors, competitively inhibits endogenous PDGF.4 Recently, the first naturally occurring antagonist for a growth factor receptor was identified in Drosophila.109 The identification of human homologs will open the way for novel approaches in molecular medicine.
Further downstream within the target cell, other modes of the inhibition of growth factor signal transduction represent a promising strategy, previously described as “signal transduction therapy.” The idea is to interrupt the growth factor–induced signal at well defined steps within the signal-transduction cascade (Fig 1⇑). A number of different compounds are currently being developed such as SH2 blockers, SH3 blockers, Ras exchange blockers, Ras farnesylation inhibitors, Raf1 blockers, and blockers of the MAPK.100 This may even allow the selective inhibition of isolated effects such as proliferation while others (eg, chemotaxis) remain intact. Gene transfer inducing the overexpression of negative regulators of the cell cycle such as active pRb11 or p2112 resulted in a reduction of neointima formation in different animal models (Table 3⇑). One has to be aware that in contrast to most of the other approaches discussed, the inhibition of the cell cycle is not a specific intervention (ie, not specific for a specific growth factor or a cell type). Such an approach does make sense, however, if the targeted cell type is well defined and a local delivery approach is successful, both of which might be realized in the prevention of restenosis. Through interference with microtubule function (an even further downstream event), taxol disrupts several growth factor–stimulated processes in the cell, such as locomotion, alteration of cell shape, and growth factor–induced proliferation. In a rat carotid artery injury model, taxol inhibits PDGF-BB–induced VSMC invasion/chemotaxis through inhibition of the PDGF-BB–induced changes in locomotion and/or shape changes, resulting in inhibition of PDGF-induced cell proliferation and neointimal accumulation of smooth muscle cells in vivo.110
Antisense oligodeoxynucleotides may be used to suppress the translation of a specific molecule,111,112 such as a growth factor, its receptor, or downstream molecules involved in mitogenic signaling. Inhibition of PDGF A-chain,113 PDGF B-chain,114 bFGF,29 or the PDGF β-receptor114 have been shown in vitro, and the effects of antisense inhibition of VEGF115 and of the PDGF β-receptor25 have recently been shown in vivo. Other successful in vivo applications of this technique have been demonstrated for downstream proliferation-associated molecules such as c-myb116 (Table 3a⇑), although there is substantial concern about the specificity of such an approach117 (see below).
Recombinant chimeric/hybrid molecules use the high binding specificity of the growth factor component and the cell-specific expression pattern of the corresponding growth factor receptors to target chemicals and drugs. Saponin linked to bFGF is able to abolish VSMC proliferation in vitro and neointima formation in vivo.118–120 Likewise, diphtheria toxin–conjugated VEGF is able to inhibit endothelial cell proliferation in vitro and neovascularization in vivo.121 These approaches are specific, potent, and clinically attractive.
A potentially useful approach for growth factor antagonism is represented by the application of angiopeptin, an octapeptide, which has been shown to inhibit neointima formation in a variety of animal models of atherosclerosis.122,123 Furthermore, there are initial, preliminary reports on its usefulness in human beings for suppression of neointima formation in transplant atherosclerosis.124 The exact mechanism of angiopeptin action is currently unknown; however, there is evidence of complex functional antagonism of IGF-1 as well as a number of other growth factors.122
Because of its different mechanism of action by signaling via membrane-bound serine/threonine kinases,15 slightly modified concepts are required for the antagonism of local TGF-β activity on the receptor and downstream-signaling level (ie, serine/threonine kinase inhibitors). In addition to the concepts discussed, there is the extracellular matrix proteoglycan decorin, a natural inhibitor of TGF-β, which binds TGF-β and was shown to function in vivo by protecting against scarring in experimental kidney disease.125
Growth Factor Agonism
Once a specific and potent function of a molecule has been established, it is a rational and straightforward approach to apply this molecule to enhance its biological activity in situations in which the naturally occurring molecule is believed to be inefficiently expressed. This can most easily be achieved through the local28 or regional52 application of the mature protein, which can be made available in large quantities with the use of recombinant DNA technology. In a number of situations, however, systemic application may be feasible.69 This is likely to be the case for the stimulation of therapeutic angiogenesis because relevant receptors are upregulated by hypoxia,48,49 leading to a response localized to the area of interest.126
Other alternatives have been developed on the basis of gene transfer technology. The transfer of a specific gene, controlled by a suitable promotor, can give rise to the production of recombinant proteins at the area of interest.127 As demonstrated in various animal models, advancements of transfer technology result in an increasing local restrictability of foreign gene expression. Depending on a variety of factors, gene therapy could be accomplished ex vivo (eg, when the vascular wall or intravascular stents are seeded with genetically modified cells)128 or in vivo through the use of different shuttles, such as retroviruses, adenoviruses, or liposomes.129 It should be emphasized that the transfection efficiency and time course of transgene expression show great variability depending on the type of vector used and the susceptibility of various cell types. As an experimental tool with which to study vessel wall biology, gene transfer experiments have already provided valuable data about the functional significance of individual factors. For example, the transfer of both PDGF-BB and TGF-β resulted in the stimulation of neointima formation in the arterial wall (Table 3b⇑). The induction of angiogenesis could be shown in different in vivo models using replication-deficient recombinant adenovirus vectors encoding the sequence of VEGF130 or FGF-5.73
Recent data give support to the idea that certain drugs may be able to enhance the expression of certain growth factors. In fact, there is first evidence from a clinical study that aspirin stimulates TGF-β activation via a so-far-unidentified pathway.131 Because elevated TGF-β levels can be correlated with a decrease in VSMC proliferation, it is conceivable that the aspirin-induced retardation of the atherosclerotic progression and the consecutive decrease in incidence of myocardial infarction132 may be explained in part (besides inhibition of platelet aggregation) by TGF-β induction. A similar effect of drug-induced TGF-β upregulation could be observed for anti-estrogens such as tamoxifen, resulting in the inhibition of VSMC proliferation in vitro133 and the suppression of diet-induced formation of lipid lesions in the mouse aorta in vivo.134
Growth Factor Therapy: Strategies and Current Limitations
Several principles are available for the inhibition or stimulation of growth factor action that have been successfully applied in the animal model to modify neointima formation or induce angiogenesis (Fig 2⇑ and Tables 3⇑ and 4⇑), whereas hematopoietic growth factors are already used in the treatment of humans. Concerning the treatment of cardiovascular diseases, the first human gene therapy trial is under way.135,136 The idea is to induce therapeutic angiogenesis in peripheral arterial occlusive disease through local application of plasmids (naked DNA) encoding VEGF (rhVEGF165). Although this is an interesting approach, a number of fundamental questions, such as the level of transgene expression, remain to be elucidated until it can be generally accepted as a rational treatment option. An alternative to intravascular DNA application could be the direct intramuscular gene transfer,137 which should even be feasible in patients with extensive peripheral vascular disease. Most recently, the first clinical phase I trials have been initiated to evaluate the feasibility of therapeutic angiogenesis in the human heart prone to coronary artery disease. The researchers will investigate the intracoronary application of recombinant bFGF (performed at the National Institutes of Health, quoted in Reference 138138 ), the application of VEGF (Genentech announcement, January 21, 1997), or the extravascular application of recombinant bFGF applied locally to the extravascular surface of a coronary artery that could not be otherwise revascularized.138
A number of unresolved questions, however, are limiting and, at the present time, prohibiting the immediate clinical use of growth factors in the treatment of cardiovascular disorders. For all the different approaches described, there are unresolved questions concerning specificity, potency, feasibility, and short- and long-term side effects in the human situation. There are general limitations to the use of antisense technology.112,139 When used under proper circumstances, this powerful tool may be applied successfully. However, recent findings point out the potential nonspecificity and lack of consistency, partly explained by an aptamer effect.140 On the other hand, there is evidence that part of the “unspecific” effect of some phosphorothioate oligodeoxynucleotides is based on their ability to directly bind the heparin-binding growth factor bFGF, consecutively preventing growth factor binding to its receptors, and therefore resulting in another mode of growth factor antagonism, which deserves further exploration.141 In addition, it was recently shown that the inhibitory effects of antisense approaches targeting c-myb and c-myc and resulting in the inhibition of neointima formation after experimental angioplasty (Table 3a⇑) are dependent on a stretch of four contiguous guanosine (G4) residues and therefore do not represent true antisense approaches.117
Immunological problems (antiglobulin response) are associated with the application of neutralizing antibodies. Therefore, attempts to humanize and modify such antibodies are under way.142 Synthetic peptides, on the other hand, vary widely in their toxic potential; some are tolerated well (such as angiopeptin124), and others are highly toxic even to cells in vitro90. Major obstacles complicating gene transfer approaches are to be clarified, including the uptake of constructs into cells, the reproducibility of recombinant gene expression, and transfer efficiency (ie, sufficient therapeutic protein levels). Moreover, minimization of virus-associated vascular pathology and the question of optimal virus composition and concentration remain issues to be resolved143,144 until a broader application in humans can be carried out reasonably and safely.
When applying or overexpressing growth factors or stimulating growth factor–dependent pathways, one should be aware of potential activation of autocrine loops, which could result in the development of cancer.2 In the case of VEGF, however, no such effect has been observed so far. In fact, VEGF did not promote transformation when overexpressed in Chinese hamster ovary cells, but it conferred a growth advantage in vivo on the basis of its angiogenic properties.145
For many approaches, a safe local delivery of the substance is sought to minimize potential side effects of the molecular intervention. In the case of intravascular applications, for example, local delivery catheters have been developed, such as double-balloon catheters, porous balloons, or devices allowing perfusion of the segment distal to the catheter tip. At the present time, however, none of the systems available allow perfect restrictability of the applied agent together with good distal perfusion of the myocardium.146 In consequence, some of the approaches discussed above cannot be undertaken at the present time—at least not without some risk of local or systemic side effects. Whether this fact will be limiting to current or future developments is presently unknown. An alternative approach aimed at the localized application of drugs to the vessel wall is the use of intravascular stents seeded with endothelial cells, which may be genetically modified and may express and secrete the protein of choice.128 Analogously, drug-coated stents may serve to target diffusible molecules to the intima and media of the vessel wall. The feasibility of this principle has been demonstrated through the use of heparin-coated stents.147
Finally, one should be aware that many growth factors have different functions in the body depending on the localization and cell type upon which they are acting. Therefore, therapeutic approaches should be as specific and restricted as possible. Only future in vivo testing will reveal the functional significance of the various actions in a given situation of molecular intervention. Nevertheless, our current technical and technological possibilities are limited, and further development in this promising field is required, for both the growth factors mentioned and other growth factors of potential importance. However, it seems only a question of time before theoretical considerations and experimental approaches regarding growth factor modulation will find their way into clinical practice.
Selected Abbreviations and Acronyms
|bFGF||=||basic fibroblast growth factor|
|MAPK||=||mitogen-associated protein kinase|
|PDGF||=||platelet-derived growth factor|
|TGF-β||=||transforming growth factor-β|
|VEGF||=||vascular endothelial growth factor|
|VSMC||=||vascular smooth muscle cells|
This work was supported in part by grants from Deutsche Forschungsgemeinschaft, Fritz-Thyssen-Stiftung, Cologne, and Dr Mildred-Scheel-Stiftung, Bonn. I thank all of my colleagues in the field for fruitful discussions. I am especially grateful to Carl-Henrik Heldin (Uppsala) and Frank Böhmer (Jena) for critically reading of the manuscript. I apologize for not having been able to cite all relevant literature because of the magnitude of available data and because the length of the reference list must be limited due to editorial policy.
- Copyright © 1997 by American Heart Association
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- Mechanisms of Growth Factor Action
- Growth Factors, Atherosclerosis, and Restenosis After Angioplasty
- Growth Factors, Myocardial Ischemia, and Angiogenesis
- Growth Factor Antagonism
- Growth Factor Agonism
- Growth Factor Therapy: Strategies and Current Limitations
- Selected Abbreviations and Acronyms
- Figures & Tables
- Info & Metrics