Molecular mechanisms of tumor angiogenesis

The maintenance of growth of malignant tumors is closely related with the development of the vascular network supplying the tumor with blood. The vascularization of tumor tissue is similar to physiological angiogenesis, but in tumors it has some specific features. During the last 25 years a vast number of biomolecules have been found and described which are involved in the regulation of tumor angiogenesis. This review considers the action mechanisms and specific features of expression of the main angiogenic growth factors, such as the vascular endothelium growth factor (VEGF), angiopoietins (Ang-1, Ang-2), and the basic fibroblast growth factor (bFGF). The roles of cytokines, growth factors, proteolytic enzymes, and cell adhesion molecules in the regulation of the key steps of blood vessel generation in the tumor are considered. The significance of angiogenesis in the treatment of oncological diseases and possible approaches for inhibition of the regulatory signals of angiogenic factors are discussed.     

Endothelial cells are activated by angiopoeitin-1 gene transfer and produce coordinated sprouting in vitro and arteriogenesis in vivo

RATIONAL AND OBJECTIVES: Activation of fully differentiated vascular cells using angiogenic genes can lead to phenotypic changes resulting in formation of new blood vessels. We tested whether Ang-1 gene transfer to endothelial cells (EC) activates these cells. METHODS AND RESULTS: EC and SMC were transduced using retroviral or adenoviral vectors to produce Ang-1 or vascular endothelial growth factor (VEGF). EC Tie-2 receptor was phosphorilated by autologous secretion of Ang-1. Transduced EC and SMC sprouting capacity was tested using collagen embedded spheroids assay and capacity to produce arteriogenesis was tested in a hind limb model of ischemia. EC expressing Ang-1 in the presence of SMC expressing VEGF exhibited high levels of sprouting of the two cell types. Flow and numbers of arteries were increased after transduced cells implantation in vivo. CONCLUSIONS: Autologous secretion of Ang-1 by transduced EC resulted in Tie-2 activation and in the presence of SMC expressing VEGF resulted in coordinated sprouting in vitro and increase in flow and number of arteries in vivo.     

Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent

In severe asthma, cytokines and growth factors contribute to the proliferation of smooth muscle cells and blood vessels, and to the increased extracellular matrix deposition that constitutes the process of airway remodeling. Vascular endothelial growth factor (VEGF), which regulates vascular permeability and angiogenesis, also modulates the function of nonendothelial cell types. In this study, we demonstrate that VEGF induces fibronectin secretion by human airway smooth muscle (ASM) cells. In addition, stimulation of ASM with VEGF activates ERK, but not p38MAPK, and fibronectin secretion is ERK dependent. Both ERK activation and fibronectin secretion appear to be mediated through the VEGF receptor flt-1, as evidenced by the effects of the flt-1-specific ligand placenta growth factor. Finally, we demonstrate that ASM cells constitutively secrete VEGF, which is increased in response to PDGF, transforming growth factor-beta, IL-1beta, and PGE(2). We conclude that ASM-derived VEGF, through modulation of the extracellular matrix, may play an important role in airway remodeling seen in asthma.     

Regulation of the expression balance of angiopoietin-1 and angiopoietin-2 by Shh and FGF-2

Sonic hedgehog (Shh) is a typical morphogen to regulate epithelial–mesenchymal interactions during embryonic development. Shh is also an indirect angiogenic agent upregulating other angiogenic factors, including angiopoietin-1 (Ang-1). Recent studies revealed that angiogenesis induced by Shh is characterized by distinct large-diameter vessels with less branching. Ang-1 promotes blood vessel maturation, and angiopoietin-2 (Ang-2) counteracts Ang-1 activity and regulates vascular branching. Thus, we hypothesized that Shh-induced angiogenesis is affected by expression of Ang-1 and Ang-2, and we investigated the regulatory system of angiopoietins by Shh in vitro. Shh enhanced Ang-1 expression but did not enhance vascular endothelial growth factor in fibroblasts. The upregulation of Ang-1 expression by Shh was significantly decreased by fibroblast growth factor-2 (FGF-2), a potent angiogenic factor. Furthermore, FGF-2 increased the expression of Ang-2 in endothelial cells. These findings suggest that Shh and FGF-2 regulate the expression balance of vascular morphogens Ang-1 and Ang-2 and are involved in angiogenesis.     

Hemochorial placentation in the primate: expression of vascular endothelial growth factor, angiopoietins, and their receptors throughout pregnancy

Vascular development and its transformation are necessary for successful hemochorial placentation, and vascular endothelial growth factor (VEGF), angiopoietins, and their receptors may be involved in the molecular regulation of this process. To determine the potential role of these putative regulators in a widely studied primate, the common marmoset, we investigated their mRNA expression and protein location in the placenta throughout pregnancy using in situ hybridization, Northern blot analysis, and immunocytochemistry. VEGF was localized in decidual and cytotrophoblast cells, and its highest expression was found in the maternal decidua. The Flt receptor was exclusively detected in the syncytial trophoblast with increasing expression in placentae from 10 wk to term. Soluble Flt (sFlt) was also detectable by Northern blot analysis. KDR receptor expression was restricted to mesenchymal cells during early placentation and to the fetoplacental vasculature during later placentation. KDR expression increased throughout pregnancy. Angiopoietin-1 (Ang-1) was localized in the syncytial trophoblast, being highly expressed in the second half of gestation. Ang-2 mRNA localized exclusively to maternal endothelial cells, and was highly expressed in 10-wk placentae. The Tie-2 receptor was found in cytotrophoblast cells and in fetal and maternal vessels. High Tie-2 levels were detected in the wall of chorion vessels at 14-wk, 17-wk, and term placentae. These results suggest that the processes of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development are regulated by the specific actions of angiogenic ligand-receptor pairs. Specifically, 1) VEGF/Flt and Ang-1/Tie-2 may promote trophoblast growth, 2) VEGF/KDR and Ang-1/Tie-2 may support fetoplacental vascular development and stabilization, 3) sFlt may balance VEGF actions, and 4) Ang-2/Tie-2 may remodel the maternal vasculature.     

VEGF and PlGF: two pleiotropic growth factors with distinct roles in development and homeostasis

Blood vessels are crucial for normal development and growth by providing oxygen and nutrients. As shown by genetic targeting studies in mice, zebrafish and Xenopus blood vessel formation (or angiogenesis) is a multistep process, which is highly dependent on angiogenic growth factors such as VEGF, the founding member of the VEGF family. VEGF binds to the tyrosine kinase receptors VEGFR-1 and VEGFR-2, and loss of VEGF or its receptors results in abnormal angiogenesis and lethality during development. In contrast, PlGF, another member of this family, binds only to VEGFR-1, and appears to be crucial exclusively for pathological angiogenesis in the adult. However, the expression of VEGFR-1 and VEGFR-2 on non-vascular cells suggests additional biological properties for these growth factors. Indeed, the VEGF family and its receptors determine development and homeostasis of many organs, including the respiratory, skeletal, hematopoietic, nervous, renal and reproductive system, independent of their vascular role. These new insights broaden the activity spectrum of these "angiogenic" growth factors, and may have therapeutic implications when using these growth factors for vascular and/or non-vascular purposes.     

Transforming growth factor-beta 1 induces angiogenesis in vitro via VEGF production in human airway smooth muscle cells

Increase in size and number of bronchial blood vessels as well as hyperaemia are factors that contribute to airway wall remodelling in patients with chronic airway diseases, such as asthma and chronic obstructive pulmonary diseases (COPD). Expression of transforming growth factor beta 1 (TGF-beta 1), a multifunctional cytokine as well as vascular endothelial growth factor (VEGF), a key angiogenic molecule, has been shown in the inflammed airways in patients with chronic airway diseases. TGF-beta 1 has been implicated in the regulation of extracellular matrix, leading to airway remodelling in patients with chronic airway diseases. However, the role of TGF-beta 1 in regulating VEGF expression in patients with chronic airway diseases, as well as the underlying mechanisms are not yet well established. We investigated whether TGF-beta 1 stimulates VEGF expression in vitro and hence could influence vascular remodelling. Cultured human airway smooth muscle cells (HASMC) were serum deprived for 60 h before incubation with 5ng/ml of TGF-beta 1 for different time points. Control cells received serum-free culture medium. TGF-beta 1 treatment resulted in time dependent HASMC cell proliferation with maximal values for DNA biosynthesis at 24 h and cell number at 48 h. Northern blot analysis of VEGF mRNA expression showed increased levels in cells treated with TGF-beta 1 for 4 to 8 h. TGF-beta 1 also induced a time-dependent release of VEGF proteins in the conditioned medium after 48 h of treatment. Furthermore, the ability of HASMC-released VEGF proteins to induce human umbilical vein endothelial cells proliferation was inhibited by VEGF receptor antagonist, confirming that TGF-beta 1 induced VEGF was biologically active. We conclude that TGF-beta 1 in addition to an extracellular matrix regulator also could play a key role in bronchial angiogenesis and vascular remodelling via VEGF pathway in asthma.     

Enhancement of bone formation by genetically-engineered bone marrow stromal cells expressing BMP-2, VEGF and angiopoietin-1

To explore the potential of combined delivery of osteogenic and angiogenic factors to bone marrow stromal cells (BMSCs) for repair of critical-size bone defects, we followed the formation of bone and vessels in tissue-engineered constructs in nude mice and rabbit bone defects upon introducing different combinations of BMP-2, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) to BMSCs with adenoviral vectors. Better osteogenesis and angiogenesis were found in co-delivery group of BMP-2, VEGF and angiopoietin-1 than any other combination of these factors in both animal models, indicating combined gene delivery of angiopoietin-1 and VEGF165 into a tissue-engineered construct produces an additive effect on BMP-2-induced osteogenesis.     

Sonic Hedgehog (Shh) Regulates the Expression of Angiogenic Growth Factors in Oxygen–Glucose-Deprived Astrocytes by Mediating the Nuclear Receptor NR2F2

Sonic hedgehog (Shh) has been found to regulate the angiogenic growth factor such as VEGF, Ang-1, and Ang-2 during ischemic insults, but the underlying mechanism is not fully understood. In this study, we employed oxygen–glucose deprivation (OGD) in astrocytes to mimic the ischemia in vitro. We found that OGD could induce the expressions of VEGF, Ang-1, and Ang-2, with the expression of Shh signaling components increased. Moreover, inhibiting the Shh signaling pathway with 5EI, a specific antibody, could decrease the expressions of VEGF, Ang-1, and Ang-2. Furthermore, the administration of exogenous Shh could induce the expressions of VEGF, Ang-1, and Ang-2 in astrocytes. The results of silencing Gli-1, or NR2F2, exhibited that exogenous Shh could regulate the expressions of VEGF, Ang-1, and Ang-2 in astrocytes by activating the NR2F2, but not the Gli-1. These results suggested that Shh could regulate the angiogenic growth factor after ischemic insults in astrocytes, and the regulation was partially mediated by the NR2F2.     

Antiangiogenic properties of natural polyphenols from red wine and green tea

Epidemiological studies have indicated that regular consumption of red wine and green tea is associated with a reduced risk of coronary heart disease and tumor progression. The development of tumors and of atherosclerosis lesions to advanced plaques, which are prone to rupture, is accelerated by the formation of new blood vessels. These new blood vessels provide oxygen and nutrients to neighboring cells. Therefore, recent studies have examined whether red wine polyphenolic compounds (RWPCs) and green tea polyphenols (GTPs) have antiangiogenic properties. In vitro investigations have indicated that RWPCs and GTPs are able to inhibit several key events of the angiogenic process such as proliferation and migration of endothelial cells and vascular smooth muscle cells and the expression of two major proangiogenic factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2, by both redox-sensitive and redox-insensitive mechanisms. Antiangiogenic properties of polyphenols have also been observed in the chick embryo chorioallantoic membrane since the local application of RWPCs and GTPs strongly inhibited the formation of new blood vessels. Moreover, intake of resveratrol or green tea has been shown to reduce corneal neovascularization induced by proangiogenic factors such as VEGF and fibroblast growth factor in mice. The ability of RWPCs and GTPs to prevent the formation of new blood vessels contributes, at least in part, to explain their beneficial effect on coronary heart disease and cancer. This review focuses on the antiangiogenic properties of natural polyphenols and examines underlying mechanisms.     

Anti-angiogenesis: A new potential strategy to inhibit restenosis

Microvessels are an integral component of the neointima developing in response to the acute vascular injury resulting from angioplasty. These vessels originate from the vasa vasorum of the adventitia, and as such appear similar to the microvessels present in atherosclerotic plaques. Several angiogenic factors have been found in atherosclerotic plaques and have been associated with increased microvascularity. In addition, most of these agents - either directly or indirectly - also induce smooth muscle cell (SMC) proliferation, an essential component of the developing neointima. We therefore propose: (1) these newly formed blood vessels are necessary for the development, maintenance, and expansion of the neointimal lesions present in restenosis; (2) the initiation, regulation and maintenance of these vessels is, at least in part, due to the coordinate sequential expression of hypoxia-inducible factor 1 (HIF-1), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), and/or other angiogenic factors such as the fibroblast growth factor (FGF) family of proteins; (3) targeted disruption of the signal transduction pathways modulated by these molecules may reduce vasa vasorum expansion and SMC proliferation. These effects, in turn, may inhibit neointimal expansion and thus the development of restenosis, especially following stenting.     

Endothelial cell survival and apoptosis in the tumor vasculature

Angiogenesis is essential for the growth and metastasis of solid tumors. The balance of endothelial cell (EC) proliferation and apoptosis is a major determinant in tumor angiogenesis. Recently, several studies demonstrated that numerous angiogenic factors not only induce angiogenesis but also function as EC survival factors. Vascular endothelial growth factor (VEGF), a potent angiogenic factor, is also an EC survival factor in embryonic vasculogenesis and tumor angiogenesis. VEGF activates specific intracellular survival pathways in ECs including Bcl-2, A1, IAP, Akt, and Erk. Integrins may function as EC survival factors by preventing anoikis by enhancing binding to the extracellular matrix. In addition, integrins may function in concert with VEGF to promote EC survival. Angiopoietin-1 (Ang-1) has recently been shown to stabilize EC networks by binding to the EC-specific tyrosine kinase receptor Tie-2. Pericytes also function as EC survival factors, by cell-cell contact, secretion of survival factors, or both. Targeting any of the above mechanisms for EC survival may provide novel antineoplastic strategies.     

Expression of vascular endothelial growth factor receptors and their ligands in rat uterus during the postpartum involution period

Abstract

Vascular endothelial growth factor (VEGF) and its specific receptors, FLt1/fms, Flk1/KDR and FLt4, play important roles in vasculogenesis, and physiological and pathological angiogenesis. Whether angiogenic growth factors are involved in regulating angiogenic processes during the postpartum involution period (PP) of the rat uterus is unknown. We used immunohistochemistry to analyze the expression levels of VEGF, the fms-like tyrosine kinase 1 (FLt1/fms), the kinase insert domain-containing region 1 (Flk1/KDR), Fms-related tyrosine kinase 4 (FLt4) and vascular endothelial growth inhibitor (VEGI) in the rat uterus during the days 1, 3, 5, 10 and 15 of the PP to determine the temporal and spatial expressions of VEGF and its receptors during the PP. Throughout the PP, cytoplasmic and membrane staining of VEGI, VEGF and their receptors were observed in the lumens, crypts and glandular epithelial cells as well as in connective tissue and vascular endothelial and smooth muscle cells in the endometrium. We found that the intensity of the immunoreactions in the endometrium varied with the morphological changes that occurred during involution. Immunoreactions for VEGI, VEGF and their receptor, Flk1/KDR, in the luminal epithelial cells were stronger than those in the glandular epithelial and stromal cells, particularly during PP 1, 3 and 5, which suggests that these peptides may contribute to re-epithelialization of the endometrium. On the other hand, Flt1/fms immunoreactivity was strong mainly in the stromal cells during the PP. The presence of VEGF and its receptors (FLt1/fms, Flk1/KDR, FLt4) in the stromal cells and blood vessels during the PP suggests that they may contribute to regulating stromal repair and angiogenesis in the involuting uterus of the rat.     

Cytokine-directed therapies for the treatment of chronic airway diseases

Multiple cytokines play a critical role in orchestrating and perpetuating inflammation in asthma and chronic obstructive pulmonary disease (COPD) and several specific cytokine and chemokine inhibitors now in development as future therapy for these diseases. Anti-IL-5 antibody markedly reduces peripheral blood and airway eosinophils, but does not appear to be effective in symptomatic asthma. Inhibition of IL-4 despite promising early results in asthma has been discontinued and blocking IL-13 might be more effective. Inhibitory cytokines, such as IL-10, interferons and IL-12 are less promising, as systemic delivery produces side effects. Inhibition of TNF-alpha may be useful in severe asthma and for treating severe COPD with systemic features. Many chemokines are involved in the inflammatory response of asthma and COPD and several small molecule inhibitors of chemokine receptors (CCR) are in development. CCR3 antagonists (which block eosinophil chemotaxis) and CXCR2 antagonists (which block neutrophil and monocyte chemotaxis) are in clinical development for asthma and COPD, respectively. Because so many cytokines are involved in asthma, drugs that inhibit the synthesis of multiple cytokines may prove to be more useful; several such classes of drug are now in clinical development and any risk of side effects with these non-specific inhibitors may be reduced by the inhaled route.     

Cell type specific expression of vascular endothelial growth factor and angiopoietin-1 and -2 suggests an important role of astrocytes in cerebellar vascularization

The vascularization of the central nervous system occurs by angiogenic sprouting, a process in which different factors like vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1/2 must act in a coordinated fashion. We investigated how these factors participate in capillarization of the cerebellum, an area experiencing marked reorganization processes during its postnatal development. VEGF and Ang-1 mRNA were predominantly expressed by astrocytes, while Ang-2 mRNA was specifically induced at the tip of invading endothelial cell cords. Similar to the cerebral cortex, vascularization of the cerebellum occurred in an inside-out pattern, following closely the generation and differentiation of each cerebellar layer. VEGF and Ang-1/2 expression patterns shifted in a similar inside-out fashion, supporting their proposed function in vessel growth and maturation.     

A broad-spectrum caspase inhibitor attenuates allergic airway inflammation in murine asthma model

Asthma is characterized by acute and chronic airway inflammation, and the severity of the airway hyperreactivity correlates with the degree of inflammation. Many of the features of lung inflammation observed in human asthma are reproduced in OVA-sensitized/challenged mice. T lymphocytes, particularly Th2 cells, are critically involved in the genesis of the allergic response to inhaled Ag. In addition to antiapoptotic effects, broad-spectrum caspase inhibitors inhibit T cell activation in vitro. We investigated the effect of the broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), on airway inflammation in OVA-sensitized/challenged mice. OVA-sensitized mice treated with z-VAD-fmk immediately before allergen challenge showed marked reduction in inflammatory cell infiltration in the airways and pulmonary blood vessels, mucus production, and Th2 cytokine production. We hypothesized that the caspase inhibitor prevented T cell activation, resulting in the reduction of cytokine production and eosinophil infiltration. Treatment with z-VAD-fmk in vivo prevented subsequent T cell activation ex vivo. We propose that caspase inhibitors may offer a novel therapeutic approach to T cell-dependent inflammatory airway diseases.