Molecular regulation of angiogenesis and lymphangiogenesis

Blood vessels and lymphatic vessels form extensive networks that are essential for the transport of fluids, gases, macromolecules and cells within the large and complex bodies of vertebrates. Both of these vascular structures are lined with endothelial cells that integrate functionally into different organs, acquire tissue-specific specialization and retain plasticity; thereby, they permit growth during tissue repair or in disease settings. The angiogenic growth of blood vessels and lymphatic vessels coordinates several biological processes such as cell proliferation, guided migration, differentiation and cell-cell communication.     

Semaphorin signaling in angiogenesis, lymphangiogenesis and cancer

Angiogenesis, the formation of new blood vessels from preexisting vasculature, is essential for many physiological processes, and aberrant angiogenesis contributes to some of the most prevalent human diseases, including cancer. Angiogenesis is controlled by delicate balance between pro- and anti-angiogenic signals. While pro-angiogenic signaling has been extensively investigated, how developmentally regulated, naturally occurring anti-angiogenic molecules prevent the excessive growth of vascular and lymphatic vessels is still poorly understood. In this review, we summarize the current knowledge on how semaphorins and their receptors, plexins and neuropilins, control normal and pathological angiogenesis, with an emphasis on semaphorin-regulated anti-angiogenic signaling circuitries in vascular and lymphatic endothelial cells. This emerging body of information may afford the opportunity to develop novel anti-angiogenic therapeutic strategies.     

Remodeling of angiogenesis and lymphangiogenesis in cervical cancer development

The ability to stimulate angiogenesis/lymphangiogenesis is an intrinsic property of cancer cells, providing them necessary conditions for growth and metastasis. The “angiogenic switch” is one of the earliest consequences of malignant transformation; it involves altered expression of numerous genes and triggers a complex set of signaling pathways in endothelial cells. Processes of tumor microvascular network formation are closely associated with the stages of carcinogenesis (from appearance of benign lesions to invasive forms) and occur with numerous deviations from the norm. Analysis of expression of proangiogenic factors during sequential steps of cervical cancer development (intraepithelial neoplasia, cancer in situ, microinvasive, and invasive cancer) provides opportunity to reconstruct the regulatory mechanisms of angiogenesis/lymphangiogenesis with emphasis on the most important components. This review summarizes literature data on expression of key regulators of angiogenesis in cervical intraepithelial neoplasia and cervical cancer and analyses their possible involvement in molecular mechanisms of neoplastic transformation of epithelial cells, as well as invasion and tumor metastasis. Correlation between expression of proangiogenic molecular factors and various clinicopathological parameters is considered in the context of their possible use in molecular diagnostics and targeted therapy of cervical cancer. Special attention is paid to rather poorly studied regulators of lymphangiogenesis and “non-VEGF dependent,” or alternative, angiogenic pathways that constitute the prospect of future research in the field.     

Notoginsenoside R1 activates the Ang2/Tie2 pathway to promote angiogenesis

BackgroundTherapeutic angiogenesis is a novel strategy for the treatment of ischemic diseases that involves promotion of angiogenesis in ischemic tissues via the use of proangiogenic agents. However, effective proangiogenic drugs that activate the Ang2/Tie2 signaling pathway remain scarce.PurposeWe aimed to investigate the proangiogenic activity of notoginsenoside R1 (NR1) isolated from total saponins of Panax notoginseng with regard to activation of the Ang2/Tie2 signaling pathway.MethodsWe examined the proangiogenic effects of NR1 by assessing the effects of NR1 on the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). The aortic ring assay and vascular endothelial growth factor receptor inhibitor (VRI)-induced vascular regression in the zebrafish model were used to confirm the proangiogenic effects of NR1 ex vivo and in vivo. Furthermore, the molecular mechanism was investigated by Western blot analysis.ResultsWe found that NR1 promoted the proliferation, mobility and tube formation of HUVECs in vitro. NR1 also increased the number of sprouting vessels in rat aortic rings and rescued VRI-induced vascular regression in zebrafish. NR1-induced angiogenesis was dependent on Tie2 receptor activation mediated by increased autocrine Ang2 in HUVECs, and inhibition of the Ang2/Tie2 pathway abrogated the proangiogenic effects of NR1.ConclusionsOur results suggest that NR1 promotes angiogenesis by activating the Ang2/Tie2 signaling pathway. Thus, NR1-induced activation of the Ang2/Tie2 pathway is an effective proangiogenic approach. NR1 may be useful agent for the treatment of ischemic diseases.     

Involvement of gamma-secretase in postnatal angiogenesis

gamma-Secretase cleaves the transmembrane domains of several integral membrane proteins involved in vasculogenesis. Here, we investigated the role of gamma-secretase in the regulation of postnatal angiogenesis using gamma-secretase inhibitors (GSI). In endothelial cell (EC), gamma-secretase activity was up-regulated under hypoxia or the treatment of vascular endothelial growth factor (VEGF). The treatment of GSI significantly attenuated growth factor-induced EC proliferation and migration as well as c-fos promoter activity in a dose-dependent manner. In vascular smooth muscle cell (VSMC), treatment of GSI significantly attenuated growth factor-induced VEGF and fibroblast growth factor-2 (FGF-2) expression. Indeed, GSI attenuated VEGF-induced tube formation and inhibited FGF-2-induced angiogenesis on matrigel in mice as quantified by FITC-lectin staining of EC. Overall, we demonstrated that gamma-secretase may be key molecule in postnatal angiogenesis which may be downstream molecule of growth factor-induced growth and migration in EC, and regulate the expression of angiogenic growth factors in VSMC.     

Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.     

Role of Angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis

This review focuses on the signaling system involving the Angiopoietin1/Tie2 receptor, which appears to be involved in the secondary stages of blood vessel formation. Although this system is crucial for blood and lymphatic vessel formation, identifying its precise role in embryonic and adult vascular biology has been a major challenge. The evidence for the key role of the Angiopoietin/Tie system is discussed, and some of the other members of the system (Ang2, Tie1) are mentioned. A comparison is made with the VEGF signaling system, which seems to provide a complementary, and somewhat more tractable, signaling system. Some of the basic unanswered questions concerning Tie/Angiopoietin biology and the secondary stages of blood vessel formation are also highlighted.     

Dual functional roles of Tie-2/angiopoietin in TNF-alpha-mediated angiogenesis

Inflammation and angiogenesis are associated with pathological disorders. TNF-alpha is a major inflammatory cytokine that also regulates angiogenesis. TNF-alpha has been shown to regulate Tie-2 and angiopoietin (Ang) expression, but the functional significance is less clear. In this study, we showed that TNF-alpha induced a weak angiogenic response in a mouse cornea assay. Systemic overexpression of Ang-1 or Ang-2 dramatically increased corneal angiogenesis induced by TNF-alpha. In the absence of TNF-alpha, neither Ang-1 nor Ang-2 promoted corneal angiogenesis. Low doses (0-25 ng/ml) of TNF-alpha increased vascular branch formation of cultured endothelial cells. Overexpression of Ang-1 or Ang-2 enhanced the effects of TNF-alpha. These data suggest that Tie-2 signaling synergistically amplifies and participates in TNF-alpha-mediated angiogenesis. In addition, high doses (>/=50 ng/ml) of TNF-alpha induced apoptosis in endothelial cells, but addition of Ang-1 or Ang-2 significantly reduced cell death. Enhanced endothelial cell survival was correlated with Akt phosphorylation. Collectively, our data reveal dual functional roles of Tie-2: low doses enhance TNF-alpha-induced angiogenesis, and high doses attenuate TNF-alpha-induced cell death. The study provides evidence supporting a role for Tie-2 in inflammatory angiogenesis.     

Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning,and only the latter role is rescued by Angiopoietin-1

VEGF and Angiopoietin-1 requisitely collaborate during blood vessel development. While Angiopoietin-1 obligately activates its Tie2 receptor, Angiopoietin-2 can activate Tie2 on some cells, while it blocks Tie2 activation on others. Our analysis of mice lacking Angiopoietin-2 reveals that Angiopoietin-2 is dispensable for embryonic vascular development but is requisite for subsequent angiogenic remodeling. Unexpectedly, mice lacking Angiopoietin-2 also exhibit major lymphatic vessel defects. Genetic rescue with Angiopoietin-1 corrects the lymphatic, but not the angiogenesis, defects, suggesting that Angiopoietin-2 acts as a Tie2 agonist in the former setting, but as an antagonist in the latter setting. Our studies define a vascular growth factor whose primary role is in postnatal angiogenic remodeling and also demonstrate that members of the VEGF and Angiopoietin families collaborate during development of the lymphatic vasculature.     

Angiopoietin-2 plays an important role in retinal angiogenesis

Angiopoietin 2 (Ang2) expression in the retina is increased during physiologic and pathologic neovascularization suggesting that it may be involved. In this study, we used Ang2-deficient mice to test that hypothesis. Mice deficient in Ang2 showed delayed and incomplete development of the superficial vascular bed of the retina, which develops primarily by vasculogenesis, and complete absence of the intermediate and deep vascular beds which develop by angiogenesis. In addition to incomplete retinal vascular development, Ang2-deficient mice showed lack of regression of the hyaloid vasculature, resulting in a phenotype that mimics infants with persistent fetal vasculature (PFV), a relatively common congenital abnormality. Exposure to high levels of oxygen resulted in partial regression of the retinal vessels, indicating that oxygen-induced regression of retinal vessels does not require Ang2. When these oxygen-exposed mice with few retinal vessels were moved to room air, there was no ischemia-induced retinal neovascularization. These data support the hypothesis that Ang2 plays a critical role in physiologic and pathologic angiogenesis, and physiologic, but not oxygen-induced vascular regression. The data also suggest that infants with PFV should be examined for genetic modifications that would be expected to cause perturbations in Tie2 signaling.     

Zebrafish and Xenopus tadpoles: small animal models to study angiogenesis and lymphangiogenesis

Small vertebrate organisms have emerged as key players in the post-genomic era for the functional characterization of novel genes on a high-throughput scale. In this context, the zebrafish embryos and Xenopus tadpoles represent attractive and valuable models to rapidly identify and characterize novel genes involved in angiogenesis and lymphangiogenesis-a significant task with a consequent impact on the design of more effective therapeutic strategies. The advantages of these two models will be discussed in the present review.     

Expression of periostin in patients with non-small cell lung cancer: correlation with angiogenesis and lymphangiogenesis

The aim of this study was to evaluate periostin expression measured immunohistochemically in patients with non-small cell lung cancer (NSCLC) and to determine its association with clinical features, prognosis, angiogenesis, and lymphangiogenesis. We investigated periostin expression in a series of 88 patients with NSCLC. We also determined whether expression of periostin correlated with microvessel density and lymphatic microvessel density. Periostin was expressed in 42% of 88 patients. Its expression was significantly correlated with tumor size, lymph node metastasis, disease stage, and lymphatic invasion (p=0.0128, 0.0015, 0.0310 and 0.0273, respectively). There also was a significant relation between periostin expression and microvessel density and lymphatic microvessel density (all p<0.0001). Five-year survival rates were better in patients with negative periostin expression than in those with positive periostin expression (p=0.0044). Periostin expression was not significant in a multivariate additive model. Our findings show that periostin correlates with increased tumor progression and a worse prognosis in NSCLC, as well as with angiogenesis and lymphangiogenesis.     

Behavior of postcapillary venule pericytes during postnatal angiogenesis.

Autogeneic bone marrow was implanted into an artificially created cavity in a segment of rat sciatic nerve, after removal of nerve fascicles, without damaging the epineurium or surrounding microcirculation. Under these conditions, the bone marrow induces capillary growth and forms granulation tissue from surrounding tissues, the behavior of pericytes being studied in the preformed (preexisting) postcapillary venules of the latter. Beginning 20 h after bone marrow implantation, the pericytes of the preexisting postcapillary venules hypertrophy, with shortening of their processes, prominent nucleoli, dispersal of ribosomes into their free form, fragmentation of basal lamina, and increased DNA synthesis. The number of contact surfaces between pericytes and endothelium is noticeably lower than in controls. Many pericytes are in mitosis. Cells with a shape transitional between pericytes and interstitial fibroblast-like cells appear. In some cases, Monastral Blue (MB) was used as a marker of the cells in preexisting venule walls of the graft bed. In the earlier stages of the experiment, the MB labelling is restricted to the cytoplasm of pericytes and endothelial cells of postcapillary venules, and to the macrophages that occur in the space between pericytes and endothelium. Furthermore, the marker continues to be observed, at a later stage, in some of the following cells: pericytes and endothelial cells of the newly formed vessels, macrophages migrating into the interstitium, transitional cells between pericytes and fibroblasts, and typical fibroblasts of the granulation tissue. The present study provides greater evidence that preformed microvasculature pericytes are substantially activated during postnatal angiogenesis and granulation tissue formation, suggesting that they may contribute to the origin of new pericytes and fibroblasts.     

The role of pericytes in angiogenesis

Pericytes are branched cells embedded within the basement membrane of capillaries and post-capillary venules. They provide an incomplete investment to endothelial cells, thus reinforcing vascular structure and regulating microvascular blood flow. Pericytes exert an important role on endothelial cell proliferation, migration and stabilization. Endothelial cells, in turn, stimulate expansion and activation of the pericyte precursor cell population. The balance between the number of endothelial cells and pericytes is highly controlled by a series of signaling pathway mechanisms operating in an autocrine and/or paracrine manner. In this review, we will first examine the molecular aspects of the pericyte activating factors secreted by endothelial cells, such as platelet derived growth factor B (PDGF-B), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β) and angiopoietins (Angs), as well as signaling pathways involving Notch and ephrins. We will then consider the complex and multivarious contribution of pericytes to the different aspects of angiogenesis with particular emphasis on the potential role of these cells as targets in tumor therapy.     

The role of adrenomedullin in angiogenesis

Adrenomedullin (AM) is a 52 amino acid peptide originally isolated from human pheochromocytoma. It was initially demonstrated to have profound effects in vascular cell biology, since AM protects endothelial cells from apoptosis, promotes angiogenesis and affects vascular tone and permeability. This review article summarizes the literature data concerning the relationship between AM and angiogenesis and describes the relationship between vascular endothelial growth factor, hypoxia and AM and tumor angiogenesis. Finally, the role of AM as a potential target of antiangiogenic therapy is discussed.