Department of Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
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ABSTRACT |
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Development of
microvascular networks is set to meet the metabolic requirements of the
tissue they perfuse. Accordingly, impairment of oxygen homeostasis,
either due to increased oxygen consumption or as a result of blood
vessel occlusion, triggers compensatory neovascularization. This
feedback reaction is mediated by a hypoxia- and hypoglycemia-induced
vascular endothelial growth factor (VEGF). VEGF accumulates under
stress as a result of increased hypoxia-inducible factor-1-mediated
transcription, stabilization of the mRNA, and the function of a
hypoxia-refractory internal ribosome entry site within its
5'-untranslated region. Matching of vascular density to the metabolic
needs of the tissue may include a process of hyperoxia-induced vessel
regression. Thus newly formed vascular networks may undergo a natural
process of vascular pruning that takes place whenever VEGF, acting as a
vascular survival factor, is downregulated below the level required to
sustain immature vessels. Immature vessels are particularly vulnerable
and are selectively obliterated upon withdrawal of VEGF. The plasticity window for vessel regression is determined by a delay in the
recruitment of periendothelial cells to the preformed endothelial
plexus. Thus fine-tuning of microvascular density takes place mostly in the newly formed plexus, but the mature system is refractory to episodic changes in tissue oxygenation. These regulatory links may
malfunction in certain pathological settings.
angiogenesis; hypoxia; hyperoxia; vessel regression
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INTRODUCTION |
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ANGIOGENESIS, defined as the formation of new blood vessels as extensions of existing vessels, in the adult, healthy organism is limited to certain organs (e.g., the female reproductive system). Remarkably, however, extensive angiogenic responses are elicited under circumstances in which oxygen homeostasis is perturbed. Thus a sustained increase in oxygen consumption or a functional impairment of existing vasculature, both resulting in insufficient perfusion, triggers neovascularization. It has long been recognized that that the driving force of compensatory angiogenesis is the condition of oxygen and nutrient deprivation. The ischemic tissue detects insufficient oxygen (hypoxia) or insufficient glucose (hypoglycemia) and responds by inducing locally the production of angiogenic factors that recruit new blood vessels to the ischemic area. The findings that the potent angiogenic factor vascular endothelial growth factor (VEGF) is induced by hypoxia and, independently, by hypoglycemia (45, 60, 64) have suggested VEGF as the molecular link in this positive feedback response. This thesis was subsequently corroborated and extended by intensive research that is reviewed below.
Adjusting the finite vascular density to match the metabolic requirements of the tissue also includes the capacity to eliminate surplus blood vessels under conditions in which oxygen supply exceeds the metabolic requirements of the tissue. Development of the retina vasculature is a prime example of a process by which a new vascular plexus is initially formed in exuberance and subsequently trimmed in an oxygen-dependent manner (4). Pruning surplus vascular loops is regarded as "economical" because it eliminates the need for long-term maintenance of unnecessary vessels. Until recently, however, the molecular mechanism underlying vascular pruning has remained unknown. Recent findings showing that VEGF functions also as a vascular survival factor essential to sustain newly formed blood vessels (2) have suggested that the levels of available VEGF might also determine the degree of vascular pruning. Studies reviewed below support the thesis that hyperoxia-induced suppression of VEGF indeed triggers the regression of surplus vessels.
It appears, therefore, that VEGF can promote both the "up-" and "downsides" in the vascular responses to imbalances in oxygenation. According to this unified view, a range of oxygen concentrations may result in the production of an "angiogenic dose," a "maintenance dose," or a "submaintenance dose" of VEGF. The review also discusses pathological circumstances of hypoxia-driven neovascularization and pathological circumstances of excessive vascular pruning.
Formation of new vascular networks by the angiogenic mode is characterized by initial formation of an endothelial cell plexus and is followed by the recruitment of periendothelial cells, namely, pericytes and smooth muscle cells. The acquisition of a periendothelial cell coating is thought to represent an important step in vessel maturation. Pertinent to the scope of this review is the thesis that "immature" and "mature" vascular networks differ with respect to their dependence on VEGF for survival. Specifically, it has been shown that immature vessels regress upon VEGF withdrawal, whereas mature vessels (operatively identified as vessels covered by periendothelial cells) appear independent of VEGF (6, 7). The existence of a transient developmental stage in which a newly formed vascular network can be remodeled to optimize perfusion rates, on one hand, and the loss of this capability at later stages, on the other hand, assures both sufficient plasticity during the formative stage and long-term stability against episodic changes in the levels of tissue oxygenation. Finally, the potential implications of enforcing the regression of immature tumor vessels are discussed.
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VEGF AS A MEDIATOR OF HYPOXIA-INDUCED ANGIOGENESIS |
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Physiological and pathological circumstances of hypoxia-induced angiogenesis. Hypoxia-induced angiogenesis is primarily an adaptive physiological response to either an increase in tissue mass (e.g., neovascularization accompanying mass increase in a muscle tissue) or a chronic increase in working load associated with elevated oxygen consumption. An increase in neuronal activity, in particular, is associated with increased oxygen consumption and usually leads to an immediate response of increased blood flow, but an intense chronic increase in neuronal activity is often accompanied by neovascularization manifested in increased vascular density (9).
At least some processes of developmental angiogenesis are driven by hypoxia. A developmental increase in oxygen consumption during organogenesis may result in a mild, subpathological level of hypoxia ("physiological hypoxia"), which may serve as a trigger for microvascular expansion. In the retina, increased oxygen consumption at the onset of photoreceptor activity results in insufficient perfusion of the overlying ganglion and inner cell layers. Hypoxia is then "sensed" by strategically located glial cells that upregulate and secrete VEGF (63). It is unlikely, however, that oxygen plays a role in stereospecific patterning of major vessels that are formed by the vasculogenic mode (49). A recent study has shown that physiological hypoxia is also essential for proper development of hematopoietic lineages (1). In the adult, observations regarding dynamic changes in vasa vasorum attest to hypoxia-induced vascular remodeling: capillaries further penetrate the muscular walls of thick vessels whenever a gap remains in the range of oxygen diffusion from the luminal and adventitial sides. Thus vasa vasorum sprouts penetrate deeper when luminal flow is compromised (e.g., at sites of atherosclerosis) and regress when flow is improved (e.g., with regression of atherosclerosis) (71, 73). Regional ischemia resulting from occlusion of blood vessels is conducive for the development of collateral blood vessels. Unlike the previous cases, collateral development is not restricted to formation of microvessels, and larger vessels are also formed. Formation of a collateral network seems to include both "classic" angiogenesis, i.e., formation of new vascular sprouts, and the recruitment of preexisting vessels. The latter still requires the formation of new connections through vascular fusions (62, 70). A third mechanism playing an important role in collateral formation is arteriogenesis (55). Arteriogenesis is distinguished by the enforcement and increase in diameter of existing vessels through interstitial growth. While it is feasible that interstitial vessel growth is also mediated by ischemia-induced factors, recent studies have highlighted the role of flow-regulated factors in arteriogenesis. According to this view, an increase in shear stress due to the diversion of flow to these vessels induces activities, other than VEGF, that directly or indirectly (e.g., through the induction of macrophage chemoattractants) lead to endothelial cell proliferation (10, 55). The adaptation of skeletal muscle in humans to endurance-type training is associated with increased capillary density and increased capillary-to-fiber ratio. Recent studies have shown that VEGF (but not basic fibroblast growth factor) is upregulated in human muscle by a single bout of dynamic exercise, thus supporting the concept that VEGF is involved in exercise-induced skeletal muscle angiogenesis (29, 47). Furthermore, a negative feedback mechanism was suggested by showing that exercise adaptation attenuates VEGF gene expression in human skeletal muscle (48). Tissue ischemia usually develops as a result of vascular injury in wounds. Hence, ischemia-driven angiogenesis is a major factor in neovascularization associated with wound healing. Indeed, a hypoxic tissue gradient is mandatory for wound-healing angiogenesis, and when the hypoxic gradient is destroyed, capillary growth ceases (35). Macrophages known to be a major source of angiogenic stimuli, including VEGF (34), are preferentially recruited to hypoxic regions of the wound (31). Ischemia-induced angiogenesis is also a major component of tumor neovascularization. Tumor angiogenesis has been traditionally viewed as the consequence of an "angiogenic switch," i.e., a genetic event that endows the tumor with the ability to recruit blood vessels from the neighboring tissue. However, tumor cells, like their normal counterparts, are able to "sense" ischemia and respond with increased angiogenesis. Therefore, stress-induced angiogenesis is an important component of tumor neovascularization, independent of angiogenic activities produced by a genetic switch. Recent studies employing tumorigenic cells deficient in their hypoxia responsiveness evaluated the relative contribution of environmental hypoxic stress to the overall angiogenic output of the tumor: hypoxia-inducible factor (HIF)-1Molecular mechanisms of hypoxic VEGF regulation.
The pivotal role that VEGF plays in mediating both vasculogenic and
angiogenic modes of blood vessel formation is reflected by the multiple
regulations of VEGF expression. Notably, various cytokines and growth
factors may regulate VEGF mRNA and protein expression, including
endothelial growth factor, transforming growth factor (TGF)-,
keratinocyte growth factor, interleukin (IL)-1
, IL-6, and
insulin-like growth factor-1 (for a recent review, see Ref.
20 and references therein). In addition, VEGF is also
subjected to hormonal regulations, including regulation by
thyroid-stimulating hormone (63) and a tight regulation by estrogens during cyclic developmental of ovarian follicles and corpus
luteum (22). A detailed account of various controls of VEGF expression, however, is beyond the scope of this review, which
focuses on its regulation by oxygen.
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VEGF AS A MEDIATOR OF VASCULAR PRUNING |
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As discussed above, the downside in the process aimed to match vascular density to changes in oxygen supply is hyperoxia-induced vascular regression. Vascular pruning takes place after the initiation of flow through the newly formed vascular network and is thought to result from excess oxygen reaching the tissue. In fact, the exposure of an immature vascular system to an experimental hyperoxia leads to an exaggerated vascular pruning and extensive elimination of many vascular loops. Notably, hyperoxia-induced obliteration of newly formed blood vessels in the retina of the premature newborn is the underlying cause of ROP. A rodent model of ROP was used to show that regression of retinal capillaries in neonatal rats exposed to high oxygen is preceded by a shutoff of VEGF production, leading in turn to selective apoptosis of endothelial cells (2). These findings have prompted the proposition that VEGF functions as a vascular survival factor and that its hyperoxic downregulation to a level lower than that required to sustain immature vessels results in their regression. This proposition was subsequently established by showing that injection of VEGF at the onset of experimental hyperoxia prevents apoptotic death of endothelial cells and rescues the retinal vasculature (2).
The most critical factor in pathogenesis of ROP, also recapitulated in the ROP rodent model, is the timing of the hyperoxic insult. Thus the incidence of ROP drops precipitously when the premature baby is placed in hyperoxia at a late gestational age (25) and does not develop in the rodent model if the insult is given later than 10 days after birth. It appears, therefore, that VEGF is critical for the survival of "immature/remodeling" blood vessels and that independence from VEGF is a molecular hallmark of maturation. Studies summarized below have shown that the recruitment of periendothelial cells to the preformed endothelial plexus renders newly formed vessels refractory to VEGF depletion, in general, and to hyperoxic insults, in particular.
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VEGF AND VESSEL MATURATION |
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Several studies have suggested that association with mural cells
is critical for proper vascular development and maintenance (for
reviews, see Refs. 5 and 15). Pericytes are cells
surrounded by basement membrane and intimately associated with
endothelial cells in the microvasculature. Pericytes express -smooth
muscle actin (
-SMA) and thus have been implied to have a contractile function. In vitro studies have highlighted the possible role of
pericytes and smooth muscle cells in regulating endothelial cell
proliferation via secretion of inhibitory growth factors such as
TGF-
(44) and inhibition of migration
(54). Supporting data for the regulation of the
endothelial cell network by pericytes is the observation that pericyte
dropout precedes proliferative retinopathy in diabetic patients
(64). A role for mural cells in maintaining vascular
integrity was suggested by a number of gene knockout studies. This
includes disruption of the genes encoding the endothelial cell-specific
receptors Tie-1 and Tie-2 (17, 46, 53), the Tie-2 ligand
angiopoietin-1 (Ang-1) (69), the tissue factor system
(13), and the platelet-derived growth factor B
(PDGF-B)/PDGF-
receptor system (36, 39). Mice deficient in these genes show a hemorrhaging phenotype often associated with a
reduced number of
-SMA-positive perivascular cells. Studies of
postnatal remodeling of the retina vasculature have shown that pericyte
recruitment proceeds by out-migration from arterioles and that coverage
of primary and smaller branches lags many days behind formation of the
endothelial plexus. Interestingly, pericyte coverage is also enhanced
by VEGF itself via an unknown mechanism (7). In vitro
studies support a direct role for VEGF in pericyte recruitment by
showing that cultured smooth muscle cells express VEGF receptors and
that VEGF acts as a smooth muscle cell chemoattractant (28). The transient existence of a pericyte-free
endothelial plexus in the retina coincides temporally and spatially
with the process of hyperoxia-induced vascular pruning. Thus the
acquisition of a pericyte coating marks the end of this plasticity
window in the retina and stabilizes its vasculature. Analysis of
angiogenic and regressive cycles in the corpus luteum has led to the
conclusion that endothelial cell survival in midstage corpus luteum is
correlated with the microvessel maturation index and that induction of
blood vessel regression during luteolysis is characterized by the
downregulation of VEGF and the upregulation of Ang-2 (26).
Pericytes are thought to provide nearby endothelial cells with
alternative survival factors, perhaps by depositing those factors in
the shared extracellular matrix.
On the basis of these findings, the following scenario is envisaged
(Fig. 1): a developmental increase in
oxygen consumption and a resultant physiological hypoxia are the
driving forces for VEGF-mediated neovascularization. However, only
after the onset of flow through the newly made vasculature can the
system be "checked" with respect to the adequacy/surplus of oxygen
reaching the tissue. Therefore, there is a physiological advantage to
maintain the option of vascular pruning for a certain time after the
formation of the initial plexus. The end of the plasticity window,
distinguished by the acquisition of a pericyte coating, prevents
deleterious vessel regression due to transient fluctuations in tissue
oxygen that may occasionally take place in the mature animal.
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This putative scenario has been documented mostly for the retina system, and it remains to be determined whether it can be generalized to include additional organs. Vascular remodeling in the postnatal brain, for example, involves vessel rarefaction (19) but may take place after the acquisition of a pericyte coating. A reduced density of arterioles and capillaries is an important common characteristic of various vascular beds in many forms of hypertension, but the mechanism of vessel rarefaction in hypertension is not clear (for review, see Ref. 68). Oxygen-induced vessel regression represents only one form of vessel regression, namely, vascular trimming taking place in temporal proximity to neovascularization. From a mechanistic point of view, it is most likely that developmentally programmed regression of fully mature functioning vascular networks (e.g., regression of hyaloid vessels in the eye) is not triggered by changes in oxygen.
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PATHOLOGICAL AND THERAPEUTIC IMPLICATIONS |
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What might be the implications of the otherwise physiological oxygen-VEGF-blood vessel connection in disease development and treatment?
The possibility that hypoxic episodes during pregnancy may contribute to congenital defects in cardiovascular development needs to be considered. Development of the cardiovascular system is exquisitely dependent on normal levels and appropriately timed expression of VEGF (12, 16, 21). Yet, VEGF is also robustly induced by environmental stresses. Hypoxic insults during embryogenesis, therefore, are likely to transiently induce high levels of VEGF, superimposed on the developmentally regulated program of expression, and, hence, contribute to congenital heart defects. The etiology of congenital heart defects (CHD) is thought to be multifactorial, involving interplay between genetic and environmental factors. The nature and mechanisms of action of the environmental insults have remained largely unknown, but the importance of embryonic hypoxia as a cardiac teratogen has long been recognized. Congenital cardiac anomalies are more prevalent at high altitude (42), and a number of studies have shown that experimental prenatal hypoxia in rats, mice, and chicks greatly increases the frequency of a host of cardiac malformations (see, e.g., Refs. 14 and 27). To date, however, the mechanism by which prenatal hypoxia contributes to CHD has not been explored.
As noted above, imbalances in both hypoxia-induced angiogenesis and hyperoxia-induced pruning are the underlying causes in the pathogenesis of major blindness-causing diseases. Specifically, pathogenesis of the different forms of retinopathy, irrespective of their etiological cause, converges at the stage of the highly ischemic retina. Already more than 50 years ago, Michaelson (43) suggested the existence of a hypoxia-inducible angiogenic factor that is responsible for the exaggerated, pathological angiogenic response. Many years later, this hypothetical factor was identified as VEGF. Current therapeutic approaches include attempts to block VEGF production or signaling as well as the inhibition of downstream events in the angiogenic cascade. In the case of ROP, which is initiated by excessive vascular pruning, the findings that administered VEGF (in its capacity as a vascular survival factor) can prevent excessive pruning have suggested a new modality for ROP prevention (2).
The prospects of therapeutic augmentation of blood delivery through
enhancement of collateral formation are exciting. Preclinical studies
have shown that collaterals can be induced in the ischemic limb
or heart through direct administration of angiogenic factors, primarily
VEGF, but also by members of the fibroblast growth factor family. Also,
different modes of angiogenic factor delivery have been used
successfully, including DNA-mediated gene transfer (for review, see
Ref. 40). These encouraging results have prompted clinical
trials that are currently underway. Induction of compensatory angiogenesis could also be attempted at different points along the
hypoxia-VEGF axis upstream of VEGF. For example, HIF-1 (or HIF-1
chimeras), which coordinates multiple homeostatic responses to hypoxia,
including the induction of VEGF, is likely to improve the perfusion of
an ischemic tissue. In principle, it might be possible to
pharmacologically induce the transduction system connecting the oxygen
sensor to upregulated target genes. Our very poor understanding of the
molecular nature of the oxygen sensor, however, has hampered this option.
In oncology, newly gained insights regarding the roles of hypoxia and VEGF in tumor biology have suggested some new approaches to keep tumor growth in check and possibly also to induce tumor regression. Preclinical studies have employed different strategies to antagonize VEGF action, including the use of anti-VEGF neutralizing antibodies, the use of soluble versions of VEGF receptors 1 and 2 acting in a dominant-negative fashion, and the design of inhibitors of the VEGF-R2 tyrosine kinase (23, 33). Encouraging positive results, manifested by a significant inhibition of tumor growth and evidence for a reduced vasculature, led to the advancement of few drugs into clinical trials. The fact that tumors generally maintain the capacity to upregulate VEGF in response to hypoxia suggests that, irrespective of the antiangiogenic method employed, vascular collapse is bound to result in extensive hypoxia that, in turn, will elicit a second angiogenic wave mediated by VEGF. Thus antagonizing VEGF might also take care of an anticipated second angiogenic wave.
To what extent is the dependence of immature blood vessels on VEGF for survival relevant to growing tumors? Tumor expansion is associated with a continuous formation of new vessels and remodeling of existing vessels. Thus, unlike blood vessels in the adjacent normal tissue, a significant fraction of tumor vessels are in a relative state of immaturity (6, 18). Immature vessels frequently found in tumors might represent a subset of vulnerable vessels requiring the continuous presence of VEGF. In fact, with the use of a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, it was shown that shutting off VEGF production leads to detachment of endothelial cells from the walls of, specifically, immature vessels and their subsequent death by apoptosis. Vascular collapse then led to hemorrhages and extensive tumor necrosis (8). Furthermore, it has been shown that early regression of vessels as a consequence of androgen-ablation therapy in prostate carcinoma is due to suppression of androgen-regulated VEGF-production (6). The dual action of VEGF as an angiogenic factor as well as a vascular survival factor may provide a mechanistic explanation to the thesis that VEGF deprivation might lead not only to inhibition of further angiogenesis but also to regression of preformed tumor vessels.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Keshet, Dept. of Molecular Biology, The Hebrew Univ.-Hadassah Medical School, Jerusalem 91120, Israel (E-mail: keshet{at}cc.huji.ac.il).
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