(Circulation. 1999;100:783-785.)
© 1999 American Heart Association, Inc.
Editorials |
Angiogenesis and Atherosclerosis
The Mandate Broadens
From Weill Medical College of Cornell University, Department of Medicine, Division of Hematology/Medical Oncology, New York, NY.
Correspondence to Roy L. Silverstein, MD, Weill Medical College of Cornell University, Department of Medicine, Division of Hematology/Medical Oncology, 1300 York Ave, Room C606, New York, NY 10021. E-mail rlsilve{at}mail.med.cornell.edu
Key Words: Editorials • angiogenesis • atherosclerosis • restenosis • cell adhesion molecules
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Introduction |
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 Top Introduction References |
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Among the most exciting developments in vascular medicine is the targeting of specific molecular regulators of blood vessel growth and development for therapeutic benefit. Angiogenesis, whether physiological, such as occurs during embryogenesis, or pathological, such as occurs during wound healing and tumor progression, requires a highly coordinated series of events that includes endothelial cell proliferation, migration, tube and lumen formation, and in some cases, recruitment of smooth muscle and other adventitial cells. A large number of studies in cellular systems and animal models have led to identification and characterization of specific factors that can either stimulate or inhibit angiogenesis.1 These studies were mainly stimulated by Folkman's hypothesis that tumor growth beyond a few millimeters requires recruitment and formation of a new microcirculation, and thus, that blood vessel–directed therapy might be effective in treating cancer. Translational research has now been initiated by several groups to test the hypothesis that local delivery of angiogenic agents, such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF), by various strategies, including viral vectors, naked DNA, or purified recombinant proteins, may improve blood flow to ischemic tissues in patients with advanced atherosclerosis. In some cases, preliminary data have been reported and appear promising.2
Specific antiangiogenic therapies are also now in the clinical-trial stage for a broadening group of common diseases, including cancer, macular degeneration, and rheumatoid arthritis. Recent studies in rodent models suggest that antiangiogenic agents may limit adipose mass in hereditary obesity and may slow progression of atheroma.3 The latter result, reported in the April 6, 1999, issue of Circulation, showed that atheromatous lesions beyond a certain size, like malignant tumors, contain an increased number of vasa vasorum and that 2 different angiogenesis inhibitors, the fumagillin analogue TNP-470 or endostatin, a proteolytic fragment of collagen XVIII, when delivered over a 16-week period, decreased lesion progression in apolipoprotein E null mice fed a cholesterol-rich "western" diet. The effect was most obvious in lesions of an intermediate age, but it was dramatic (70% to 85% reduction in plaque area). These studies substantiate the hypothesis published 15 years ago that neovascularization by vasa vasorum in coronary vessels contributes to atherogenesis.4
In the current issue of Circulation, Chen et al5 have identified a new therapeutic target for vascular disease that may relate to the same biological regulatory mechanisms that control angiogenesis. They show that a specific neutralizing monoclonal antibody directed against thrombospondin-1 (TSP-1) improved "healing" of a balloon-injured rat carotid artery. The antibody was delivered locally into the lumen at the time of the injury and then by constant infusion into the vessel wall adventitia for 2 to 4 weeks. The authors documented successful delivery of the antibody into the vessel wall by immunohistochemical techniques and demonstrated a statistically significant increased rate of reendothelialization in the treated arteries along with a decrease in the ratio of intima-to-media thickness. These effects were associated with an increased number of proliferating cells at the lumen (presumably migrating endothelial cells) and a decreased number of proliferating cells in the media (presumably smooth muscle cells).
So what is TSP-1, and why should targeting this molecule lead to
accelerated reendothelialization and reduced
neointima formation? TSP-1 is a 450 000-Da matrix
glycoprotein6 of long-standing interest to
vascular biologists because of its pattern of expression and its
ability to modulate many important vascular cell functions. It is
present in large amounts in platelet 
-granules, from which
it is secreted at sites of platelet activation. It is also made and
secreted by endothelial cells and smooth muscle cells
in a highly regulated manner. Its expression is rapidly and
dramatically upregulated in response to vascular injury or exposure of
cells to platelet-derived growth factor (PDGF) or bFGF; thus, it is
present in large amounts in atheroma and in the matrix
of a balloon-injured vessel. Of most relevance to angiogenesis and
repair of arterial balloon injury are the observations that
TSP-1 is a highly potent inhibitor of angiogenesis in vivo
and is capable of blocking capillary endothelial cell
proliferation, migration, and tube formation induced by angiogenic
factors.7 It also enhances smooth muscle proliferation and
migration in response to PDGF.6 Antibodies to TSP-1 thus
could potentially augment the endothelial cell
migration and proliferation necessary for
reendothelialization while blocking the smooth muscle
cell migration and proliferation associated with accelerated
restenosis.
The biology of TSP-1, however, is complex, and the effects seen in
response to delivery of anti–TSP-1 into an injured vessel are likely
to be much more complicated than those described above. TSP-1 interacts
with many matrix constituents,6 including heparan sulfate
proteoglycans, fibronectin, and collagen, and probably plays a role in
matrix stability and remodeling. Evidence from TSP-1 null mice suggests
that this is particularly important in bone matrix, but the role of
TSP-1 in vascular wound matrix remodeling and stability has not yet
been well characterized. TSP-1 can also interact with several specific
cellular adhesion receptors, including CD36,8
v-integrins,9 heparan sulfate
proteoglycans, and integrin-associated protein (IAP;
CD47).10 It can function as an adhesion molecule,
promoting tumor cell–matrix interactions9 and
platelet aggregation, but it also has antiadhesive properties for
some cells, leading, for example, to disruption of
endothelial cell focal adhesion plaques. On
macrophages, TSP-1 mediates recognition and phagocytosis of
apoptotic leukocytes, thus participating in the later stages of
the inflammatory response and limiting proinflammatory
influences.11 TSP-1 interacts with several proteases,
including plasminogen, urokinase, thrombin, cathepsin, and
elastase, promoting plasmin generation6 but inhibiting
cathepsin. It also binds transforming growth factor-ß (TGF-ß) with
high affinity and can efficiently convert the latent form into the
active form.12 This latter effect is probably very
important in vascular injury responses. TGF-ß is a highly active
cytokine and, like TSP-1, is also found in large amounts in
injured vessels. It promotes collagen production and matrix
deposition by vascular cells, regulates plasmin generation, and has
many important anti-inflammatory properties. Studies of the TGF-ß
null mouse and the TSP-1 null mouse have shown, in fact, that the 2
phenotypes are similar and that TSP-1 is the critical in vivo
activator of TGF-ß.12
Two critical questions arise from these observations. How can a single
molecule effect so many disparate functions, and to which of the many
functions attributable to TSP-1 can the "healing" effects of
anti–TSP-1 treatment be ascribed? The answer to the first question is
the most straightforward and relates directly to the structural
organization of the protein. TSP-1 is a homotrimeric protein
structurally organized as a series of discrete modules in linear
array6 (Figure
). The most
N-terminal of these modules is a domain responsible for heparin binding
and for disulfide bond–dependent trimerization of the TSP monomers.
Immediately adjacent to the heparin binding domain is a cysteine-rich
region with homology to procollagen. After that are 3 copies of a
domain called the type I repeat, sequences of 50 to 54 amino acids that
are homologous to malaria proteins and properdin. Adjacent to this
region are 3 copies of type II repeats bearing epidermal growth
factor–like homology, and after that are 7 type III repeats
bearing homology to calcium binding sites in many other proteins. These
are followed by a unique globular carboxy-terminal domain. Studies
using protease digestion, monoclonal antibodies, synthetic peptides,
and recombinant TSP-1 fragments have shown that each of these domains
is responsible for specific functions. Thus, the apparently
contradictory studies showing that TSP-1 has both proadhesive and
antiadhesive properties are explained by the existence of multiple
TSP-1 cellular adhesion receptors, each with specificity for a
different TSP domain (Figure) and each with its own unique
pattern of cellular expression. The TSP type I repeats contain the
amino acid sequence CSVTCG, which has been shown to mediate binding to
CD36.13 The last type III repeat contains the integrin
binding domain, RGDA,9 and the carboxy-terminal domain
contains the binding site for IAP.10 The TGF-ß binding
and activation domain also appears to be in the type I repeats, but it
is not known whether it is identical to the CSVTCG sequence required
for CD36 binding.
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The CD36–TSP-1 interaction is particularly relevant to angiogenesis
and vascular biology. CD36 is an 88 000-Da
glycoprotein expressed on platelets,
macrophages, adipocytes, and certain specialized epithelia
(retinal pigment and breast).14 15 It too is a complex
multifunctional protein, serving as a scavenger receptor on
macrophages for oxidized LDL and apoptotic
cells16 17 and as a fatty acid transporter on adipocytes
and muscle.18 On endothelium, its
expression is limited to microvessels, the vessels from which new
vessels arise during an angiogenic response. CD36 is the receptor that
mediates some of the antiangiogenic properties of TSP-1. Studies by
Bouck and colleagues, collaborating with our group,19 have
shown that blockade of the TSP-1–CD36 interaction with recombinant
CD36 peptides or inhibitory antibodies blocked the ability
of TSP-1 to inhibit endothelial cell proliferation,
migration, and tube formation. Synthetic peptides from the type I TSP
repeat or from related sequences in other molecules have been shown to
bind CD36 and to inhibit angiogenesis. Furthermore, transfection of
CD36 cDNA into TSP-1–unresponsive macrovascular
endothelial cells conferred responsiveness to TSP-1 in
in vitro angiogenesis assays. Studies on a CD36 null mouse generated in
our laboratory have shown that angiogenesis is not inhibited by TSP-1
in an in vivo corneal angiogenesis assay. A second region of the TSP-1
molecule from the procollagen homology domain also has antiangiogenic
activity, implicating TSP-1–matrix interactions in angiogenesis as
well. Although v-integrins have been
implicated in endothelial cell survival and
apoptosis, the integrin binding region of TSP-1 has not been
shown to have antiangiogenic activity.
The question of which of the many functions attributable to TSP-1 is responsible for the healing effects of anti–TSP-1 treatment is a more difficult one. There are 2 pieces of evidence suggesting that CD36 might not be involved. If, in fact, the cells responsible for reendothelialization are migrating in from the periphery of the injury, they are not likely to express CD36. Macrovascular endothelial cells, such as in the carotid artery, are CD36 negative. Second, the monoclonal antibody used by Chen et al,5 C6.7, recognizes the carboxy-terminal IAP binding domain on TSP-1, not the CD36 binding region in the type I repeats. IAP is a membrane-spanning protein shown to modulate integrin function and signaling on vascular cells.10 C6.7 can block chemotaxis and spreading of endothelial cells on RGD-containing substrates, presumably by blocking the ability of TSP-1 to mediate IAP-dependent integrin activation. On the other hand, recent studies20 suggest the existence of a circulating pool of endothelial cell progenitors with high angiogenic potential. If reendothelialization is mediated in part by homing of these cells to the lesion, with subsequent proliferation and differentiation, it is possible that TSP could function by blocking these phenomena in a CD36-dependent manner. Also, although C6.7 does not recognize the type I repeat, it might interfere with CD36 binding and function by steric effects. Thus, although the studies by Chen et al5 are intriguing, significantly more characterization at the molecular level will be required to determine whether the observed effects are due to antiendothelial proliferation/migration activities, pro–smooth muscle cell proliferation activities, TGF-ß activation, integrin interactions, IAP interactions, matrix stability, protease activity, or even effects on macrophage activation.
The provocative preliminary studies published in Circulation this year by Chen et al5 and Moulton et al3 may help in defining the precise clinical scenarios suitable for proangiogenesis- or antiangiogenesis-based therapies for patients with atherosclerosis. The use of antiangiogenic therapies to halt progression of lesions is exciting and logical but may be limited by a restricted temporal window of opportunity and, in certain circumstances such as angioplasty, may be associated with exacerbating restenosis after balloon angioplasty. The use of proangiogenic factors as an in vivo bypass strategy is equally logical and is supported by intriguing preliminary data. On the basis of studies similar to the one reported by Chen et al,5 this strategy might also be of benefit for the angiogenesis-related process of reendothelialization of a balloon-injured artery. In this regard, TSP-1 might be an attractive therapeutic target because of its additional effects on smooth muscle cells, TGF-ß, and the inflammatory response, which could limit the exaggerated proliferative responses in the vessel wall that lead to accelerated restenosis. The caveats of these studies include the potential risk of promoting plaque instability and lesion progression if, as suggested by Barger et al4 and others and as supported by the recent studies of Moulton et al,3 neovascularization in the lesion is proatherosclerotic. It is possible that as we learn more about the mechanisms of the arterial healing effects of TSP-1, "designer" molecules based on structure/function relationships could be developed to maximize the therapeutic effects of TSP-1 while minimizing potential negative effects.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionReferences |
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