Growth regulation of the vascular system: an emerging role for adenosine

The importance of metabolic factors in the regulation of angiogenesis is well understood. An increase in metabolic activity leads to a decrease in tissue oxygenation causing tissues to become hypoxic. The hypoxia initiates a variety of signals that stimulate angiogenesis, and the increase in vascularity that follows promotes oxygen delivery to the tissues. When the tissues receive adequate amounts of oxygen, the intermediate effectors return to normal levels, and angiogenesis ceases. An emerging concept is that adenosine released from hypoxic tissues has an important role in driving the angiogenesis. The following feedback control hypothesis is proposed: AMP is dephosphorylated by ecto-5'-nucleotidase, producing adenosine under hypoxic conditions in the extracellular space adjacent to a parenchymal cell (e.g., cardiomyocyte, skeletal muscle fiber, hepatocyte, etc.). Extracellular adenosine activates A(2) receptors, which stimulates the release of vascular endothelial growth factor (VEGF) from the parenchymal cell. VEGF binds to its receptor (VEGF receptor 2) on endothelial cells, stimulating their proliferation and migration. Adenosine can also stimulate endothelial cell proliferation independently of VEGF, which probably involves modulation of other proangiogenic and antiangiogenic growth factors and perhaps an intracellular mechanism. In addition, hemodynamic factors associated with adenosine-induced vasodilation may have a role in the development and remodeling of the vasculature. Once a new capillary network has been established, and the diffusion/perfusion capabilities of the vasculature are sufficient to supply the parenchymal cells with adequate amounts of oxygen, adenosine and VEGF as well as other proangiogenic and antiangiogenic growth factors return to near-normal levels, thus closing the negative feedback loop. The available data indicate that adenosine might be an essential mediator for up to 50-70% of the hypoxia-induced angiogenesis in some situations; however, additional studies in intact animals will be required to fully understand the quantitative importance of adenosine.     

Regulation of retinal capillary cells by basic fibroblast growth factor, vascular endothelial growth factor, and hypoxia

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) feature prominently in retinal neovascular diseases. Although the role of VEGF in retinal angiogenesis is well established, the importance of bFGF in this process requires further clarification. This study was undertaken to investigate the responses of retinal capillary cells (endothelial cells and pericytes) to bFGF under hypoxic conditions, as well as the potentially synergistic effects of bFGF and VEGF on the proliferation and cord formation of retinal endothelial cells. Cell proliferation was determined by cell number and by 3H-thymidine incorporation. Cord formation was assessed in three-dimensional gels of collagen type I. VEGF and bFGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced in a hypoxic environment. Moreover, the proliferation of pericytes was stimulated to a greater extent by bFGF relative to VEGF. Endothelial migration in collagen gels, however, was induced more effectively by VEGF than by bFGF. A synergistic effect of VEGF and bFGF on cell invasion was observed in the collagen gel assay. VEGF and bFGF each augment proliferation of these cells, especially under hypoxia. We thus propose that these two cytokines have a synergistic effect at several stages of angiogenesis in the retina.     

Hypoxia associated NMDA receptor 2 subunit composition: developmental comparison between the hypoxia-tolerant subterranean mole-rat, Spalax, and the hypoxia-sensitive rat

Vertebrate brains are sensitive to oxygen depletion, which may lead to cell death. Hypoxia sensitivity originates from the high intrinsic rate of ATP consumption of brain tissue, accompanied by the release of glutamate, leading to the opening of ionotropic glutamate receptors, such as N-methyl-D-aspartate (NMDA) receptors (NMDARs). The relative expression levels of the four NMDAR-2 (NR2) subunits change during mammalian development with higher levels of units NR2B and NR2D observed during early development and correlated with hypoxic tolerance during embryonic and neonatal stages of development. Higher levels of NR2D are also abundant in brains of hypoxia tolerant species such as the crucian carp. The subterranean mole-rat, Spalax spends its life underground in sealed burrows and has developed a wide range of adaptations to this special niche including hypoxia-tolerance. In this study, we compared the in vivo mRNA expression of NR2 subunits in the brains of embryonic, neonatal and adult Spalax and rat. Our results demonstrate that under normoxic conditions, mRNA levels of NR2D are higher in Spalax than in rat at all developmental stages studied and are similar to levels in neonatal rat and in other hypoxia/anoxia tolerant species. Furthermore, under hypoxia Spalax NR2D mRNA levels increase while no response was observed in rat. Similarly, hypoxia induces an increase in mRNA levels of Spalax NR2A, claimed to promote neuronal survival. We suggest that indeed the proportional combinations of NMDAR-2 subunits contribute to the ability of the Spalax brain to cope with hypoxic environments.     

Reduced O(2) concentration during CAM development - Its effect on angiogenesis and gene expression in the broiler embryo CAM

Hypoxia during embryogenesis may induce changes in the development of some physiological regulatory systems, thereby causing permanent phenotypic changes in the embryo. Various levels of hypoxia at different time points during embryogenesis were found to affect both anatomical and physiological morphogenesis. These changes and adaptations depended on the timing, intensity, and duration of the hypoxic exposure and, moreover, were regulated by differential expression of developmentally important genes, mostly expressed in a stage- and time-dependent manner. Eggs incubated in a 17%-oxygen atmosphere for 12h/d from E5 through E12 exhibited a clear and significant increase in the vascular area of the chorioallantoic membrane (CAM); an increase that was already significant within 12h after the end of the 1st hypoxic exposures (E6). We used the combination of the genes, β-actin, RPLP0 and HPRT as a reference for gene expression profiling, in studying the expression levels of hypoxia-inducible factor 1-alpha (HIF1α), vascular endothelial growth factor alpha-2 (VEGF α 2), vascular endothelial growth factor receptor 2 (KDR), matrix metalloproteinase-2 (MMP2), and fibroblast growth factor 2 (FGF2), under normal and hypoxic conditions. In general, expression of all five investigated genes throughout the embryonic day of development had similar patterns of hypoxia-induced alterations. In E5.5 embryos, expression of HIF1α, MMP2, VEGFα2, and KDR was significantly higher in hypoxic embryos than in controls. In E6 embryos expression of HIF1α, VEGFα2, and FGF2 was significantly higher in hypoxic embryos than in controls. From E6.5 onward expression levels of the examined genes did not show any differences between hypoxic and control embryos. It can be concluded that in this experimental model, exposing broiler embryos to 17% O(2) from E5 to E7 induced significant angiogenesis, as expressed by the above genes. Further studies to examine whether this early exposure to hypoxic condition affects the chick's ability to withstand a post-hatch hypoxic environment is still required.     

高原鼢鼠血管内皮生长因子基因编码和mRNA的表达以及微血管密度:与其它鼠类的比较

动物组织微血管密度(microvessel density,MVD)的大小与其对低氧的适应能力有关.为进一步探讨高原鼢鼠对严重低氧、高CO2洞道环境的适应机制,本文就高原鼢鼠脑组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)的mRNA表达水平及MVD与其它鼠类进行...     

Myocardial Hypertrophy Overrides the Angiogenic Response to Hypoxia

Background

Cyanosis and myocardial hypertrophy frequently occur in combination. Hypoxia or cyanosis can be potent inducers of angiogenesis, regulating the expression of hypoxia-inducible factors (HIF), vascular endothelial growth factors (VEGF), and VEGF receptors (VEGFR-1 and 2); in contrast, pressure overload hypertrophy is often associated with impaired pro-angiogenic signaling and decreased myocardial capillary density. We hypothesized that the physiological pro-angiogenic response to cyanosis in the hypertrophied myocardium is blunted through differential HIF and VEGF-associated signaling.

Methods and Results

Newborn rabbits underwent aortic banding and, together with sham-operated littermates, were transferred into a hypoxic chamber (FiO₂ = 0.12) at 3 weeks of age. Control banded or sham-operated rabbits were housed in normoxia. Systemic cyanosis was confirmed (hematocrit, arterial oxygen saturation, and serum erythropoietin). Myocardial tissue was assayed for low oxygen concentrations using a pimonidazole adduct. At 4 weeks of age, HIF-1α and HIF-2α protein levels, HIF-1α DNA-binding activity, and expression of VEGFR-1, VEGFR-2, and VEGF were determined in hypoxic and normoxic rabbits. At 6 weeks of age, left-ventricular capillary density was assessed by immunohistochemistry. Under normoxia, capillary density was decreased in the banded rabbits compared to non-banded littermates. As expected, non-hypertrophied hearts responded to hypoxia with increased capillary density; however, banded hypoxic rabbits demonstrated no increase in angiogenesis. This blunted pro-angiogenic response to hypoxia in the hypertrophied myocardium was associated with lower HIF-2α and VEGFR-2 levels and increased HIF-1α activity and VEGFR-1 expression. In contrast, non-hypertrophied hearts responded to hypoxia with increased HIF-2α and VEGFR-2 expression with lower VEGFR-1 expression.

Conclusion

The participation of HIF-2α and VEGFR-2 appear to be required for hypoxia-stimulated myocardial angiogenesis. In infant rabbit hearts with pressure overload hypertrophy, this pro-angiogenic response to hypoxia is effectively uncoupled, apparently in part due to altered HIF-mediated signaling and VEGFR subtype expression.     

Skeletal muscle capillarity and angiogenic mRNA levels after exercise training in normoxia and chronic hypoxia.

Gene expression of vascular endothelial growth factor (VEGF), and to a lesser extent of transforming growth factor-beta(1) (TGF-beta(1)) and basic fibroblast growth factor (bFGF), has been found to increase in rat skeletal muscle after a single exercise bout. In addition, acute hypoxia augments the VEGF mRNA response to exercise, which suggests that, if VEGF is important in muscle angiogenesis, hypoxic training might produce greater capillary growth than normoxic training. Therefore, we examined the effects of exercise training (treadmill running at the same absolute intensity) in normoxia and hypoxia (inspired O(2) fraction = 0.12) on rat skeletal muscle capillarity and on resting and postexercise gene expression of VEGF, its major receptors (flt-1 and flk-1), TGF-beta(1), and bFGF. Normoxic training did not alter basal or exercise-induced VEGF mRNA levels but produced a modest twofold increase in bFGF mRNA (P < 0.05). Rats trained in hypoxia exhibited an attenuated VEGF mRNA response to exercise (1.8-fold compared 3.4-fold with normoxic training; P < 0.05), absent TGF-beta(1) and flt-1 mRNA responses to exercise, and an approximately threefold (P < 0.05) decrease in bFGF mRNA levels. flk-1 mRNA levels were not significantly altered by either normoxic or hypoxic training. An increase in skeletal muscle capillarity was observed only in hypoxically trained rats. These data show that, whereas training in hypoxia potentiates the adaptive angiogenic response of skeletal muscle to a given absolute intensity of exercise, this was not evident in the gene expression of VEGF or its receptors when assessed at the end of training.     

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40ng/ml in the culture medium, but decreased at 80ng/ml. Under CoCl(2)-induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or anti-angiogenic activities of BMSCs.     

Exercise training improves aging-induced downregulation of VEGF angiogenic signaling cascade in hearts

Exercise training improves aging-induced deterioration of angiogenesis in the heart. However, the mechanisms underlying exercise-induced improvement of capillary density in the aged heart are unclear. Vascular endothelial growth factor (VEGF) is implicated in angiogenesis, which activated angiogenic signaling cascade through Akt and endothelial nitric oxide synthase (eNOS)-related pathway. We hypothesized that VEGF angiogenic signaling cascade in the heart contributes to a molecular mechanism of exercise training-induced improvement of capillary density in old age. With the use of hearts of sedentary young rats (4 mo old), sedentary aged rats (23 mo old), and exercise-trained aged rats (23 mo old, swim training for 8 wk), the present study investigated whether VEGF and VEGF-related angiogenic molecular expression in the aged heart is affected by exercise training. Total capillary density in the heart was significantly lower in the sedentary aged rats compared with the sedentary young rats, whereas that in the exercise-trained rat was significantly higher than the sedentary aged rats. The mRNA and protein expressions of VEGF and of fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk-1), which are main VEGF receptors, in the heart were significantly lower in the sedentary aged rats compared with the sedentary young rats, whereas those in the exercise-trained rats were significantly higher than those in the sedentary aged rats. The phosphorylation of Akt protein and eNOS protein in the heart corresponded to the changes in the VEGF protein levels. These findings suggest that exercise training improves aging-induced downregulation of cardiac VEGF angiogenic signaling cascade, thereby contributing to the exercise training-induced improvement of angiogenesis in old age.     

Angiogenesis and vascular remodelling in normal and cancerous tissues

Vascular development and homeostasis are underpinned by two fundamental features: the generation of new vessels to meet the metabolic demands of under-perfused regions and the elimination of vessels that do not sustain flow. In this paper we develop the first multiscale model of vascular tissue growth that combines blood flow, angiogenesis, vascular remodelling and the subcellular and tissue scale dynamics of multiple cell populations. Simulations show that vessel pruning, due to low wall shear stress, is highly sensitive to the pressure drop across a vascular network, the degree of pruning increasing as the pressure drop increases. In the model, low tissue oxygen levels alter the internal dynamics of normal cells, causing them to release vascular endothelial growth factor (VEGF), which stimulates angiogenic sprouting. Consequently, the level of blood oxygenation regulates the extent of angiogenesis, with higher oxygenation leading to fewer vessels. Simulations show that network remodelling (and de novo network formation) is best achieved via an appropriate balance between pruning and angiogenesis. An important factor is the strength of endothelial tip cell chemotaxis in response to VEGF. When a cluster of tumour cells is introduced into normal tissue, as the tumour grows hypoxic regions form, producing high levels of VEGF that stimulate angiogenesis and cause the vascular density to exceed that for normal tissue. If the original vessel network is sufficiently sparse then the tumour may remain localised near its parent vessel until new vessels bridge the gap to an adjacent vessel. This can lead to metastable periods, during which the tumour burden is approximately constant, followed by periods of rapid growth.     

VEGF deprivation-induced apoptosis is a component of programmed capillary regression

The pupillary membrane (PM) is a transient ocular capillary network, which can serve as a model system in which to study the mechanism of capillary regression. Previous work has shown that there is a tight correlation between the cessation of blood flow in a capillary segment and the appearance of apoptotic capillary cells throughout the segment. This pattern of cell death is referred to as synchronous apoptosis (Lang, R. A., Lustig, M., Francois, F., Sellinger, M. and Plesken, H. (1994) Development 120, 3395-3404; Meeson, A., Palmer, M., Calfon, M. and Lang, R. A. (1996) Development 122, 3929-3938). In the present study, we have investigated whether the cause of synchronous apoptosis might be a segmental deficiency of either oxygen or a survival factor. Labeling with the compound EF5 in a normal PM indicated no segmental hypoxia; this argued that oxygen deprivation was unlikely to be the cause of synchronous apoptosis. When rat plasma was used as a source of survival factors in an in vitro PM explant assay, inhibition of vascular endothelial growth factor (VEGF) all but eliminated the activity of plasma in suppressing apoptosis. This argued that VEGF was an important plasma survival factor. Furthermore, inhibition of VEGF in vivo using fusion proteins of the human Flk-1/KDR receptor resulted in a significantly increased number of capillaries showing synchronous apoptosis. This provides evidence that VEGF is necessary for endothelial cell survival in this system and in addition, that VEGF deprivation mediated by flow cessation is a component of synchronous apoptosis.     

Characterization of vascular endothelial growth factor receptors on the endothelial cell surface during hypoxia using whole cell binding arrays

Angiogenesis plays a central role in a variety of important biological processes such as reproduction, tissue development, and wound healing, as well as being critical to tumor formation in cancer. The development of chromosomal substitution (consomic) rat strains has permitted the chromosomal localization of genetic factors critical to angiogenesis, but many questions remain as to the mechanisms involved. Here we utilize a novel cell capture assay to assess changes in the functional expression of vascular endothelial growth factor (VEGF) receptors on the surface of vascular endothelial cells isolated from rat strains that are normal or impaired in angiogenesis. We show that functional VEGF receptor expression is increased under hypoxic conditions in rat strains that exhibit normal angiogenesis but not in a strain impaired in angiogenesis. This result implicates the dysregulation of VEGF receptor expression levels on the endothelial cell surface as a key factor in impaired angiogenesis.     

Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium

We hypothesised that angiopoietin-1 (Ang-1), in conjunction with vascular endothelial growth factor (VEGF) gene therapy, can enhance arteriogenesis and angiogenesis during myocardial ischemia. Mice were given a single intramyocardial injection of saline, phVEGF-A(165) and phAng-1 or a combination thereof into the non-ischemic normal heart or into the ischemic border zone of the infarcted heart. In the normal and the ischemic myocardium, gene transfer of phVEGF-A(165) alone increased the myocardial capillary density by 16% and 36%, respectively, and phAng-1 had a similar effect. In the normal heart, the ratio of arteriolar to capillary densities increased with phVEGF-A(165) and more so in the ischemic myocardium where phAng-1 also had an effect. Furthermore, the combination of plasmids induced an up to 7.5-fold increase. Transient overexpression of VEGF-A(165) boosts endogenous arteriogenesis in addition to capillary angiogenesis. Ang-1 further boosts this effect at the arteriolar level.