Carolyn A. Staton

A critical analysis of current in vitro and in vivo angiogenesis assays

The study of angiogenesis has grown exponentially over the past 40 years with the recognition that angiogenesis is essential for numerous pathologies and, more recently, with the advent of successful drugs to inhibit angiogenesis in tumours. The main problem with angiogenesis research remains the choice of appropriate assays to evaluate the efficacy of potential new drugs and to identify potential targets within the angiogenic process. This selection is made more complex by the recognition that heterogeneity occurs, not only within the endothelial cells themselves, but also within the specific microenvironment to be studied. Thus, it is essential to choose the assay conditions and cell types that most closely resemble the angiogenic disease being studied. This is especially important when aiming to translate data from in vitro to in vivo and from preclinical to the clinic. Here we critically review and highlight recent advances in the principle assays in common use including those for endothelial cell proliferation, migration, differentiation and co‐culture with fibroblasts and mural cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as chick chorioallantoic membrane (CAM), zebrafish, sponge implantation, corneal, dorsal air sac, chamber and tumour angiogenesis models. Finally, we briefly discuss the direction likely to be taken in future studies, which include the use of increasingly sophisticated imaging analysis systems for data acquisition.

Current methods for assaying angiogenesis in vitro and in vivo

Angiogenesis, the development of new blood vessels from an existing vasculature, is essential in normal developmental processes and in numerous pathologies, including diabetic retinopathy, psoriasis and tumour growth and metastases. One of the problems faced by angiogenesis researchers has been the difficulty of finding suitable methods for assessing the effects of regulators of the angiogenic response. The ideal assay would be reliable, technically straightforward, easily quantifiable and, most importantly, physiologically relevant. Here, we review the advantages and limitations of the principal assays in use, including those for the proliferation, migration and differentiation of endothelial cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as sponge implantation, corneal, chamber, zebrafish, chick chorioallantoic membrane (CAM) and tumour angiogenesis models.

Neuropilins in physiological and pathological angiogenesis

Neuropilin‐1 (Np1) and neuropilin‐2 (Np2) are transmembrane glycoproteins with large extracellular domains that interact with both class 3 semaphorins and vascular endothelial growth factor (VEGF), and are involved in the regulation of many physiological pathways, including angiogenesis. The neuropilins also interact directly with the classical receptors for VEGF, VEGF‐R1 and ‐R2, mediating signal transduction. The heart, glomeruli and osteoblasts express both Np1 and Np2, but there is differential expression in the adult vasculature, with Np1 expressed mainly by arterial endothelium, whereas Np2 is only expressed by venous and lymphatic endothelium. Both neuropilins are commonly over‐expressed in regions of physiological (wound‐healing) and pathological (tumour) angiogenesis, but the signal transduction pathways, neuropilin‐mediated gene expression and the definitive role of neuropilins in angiogenic processes are not fully characterized. This review details the current evidence for the role of neuropilins in angiogenesis, and suggests future research directions that may enhance our understanding of the mechanisms of action of this unique family of proteins. Copyright

Classification and functions of enteroendocrine cells of the lower gastrointestinal tract

With over thirty different hormones identified as being produced in the gastrointestinal (GI) tract, the gut has been described as ‘the largest endocrine organ in the body’ (Ann. Oncol., 12, 2003, S63). The classification of these hormones and the cells that produce them, the enteroendocrine cells (EECs), has provided the foundation for digestive physiology. Furthermore, alterations in the composition and function of EEC may influence digestive physiology and thereby associate with GI pathologies. Whilst there is a rapidly increasing body of data on the role and function of EEC in the upper GI tract, there is a less clear‐cut understanding of the function of EEC in the lower GI. Nonetheless, their presence and diversity are indicative of a role. This review focuses on the EECs of the lower GI where new evidence also suggests a possible relationship with the development and progression of primary adenocarcinoma.

The angiogenic switch occurs at the adenoma stage of the adenoma-carcinoma sequence in colorectal cancer

Objective: The aim of this study was to examine the relationship between tissue factor (TF), vascular endothelial growth factor (VEGF) and the onset of angiogenesis in the adenoma–carcinoma sequence (ACS), the stepwise process encompassing colorectal cancer (CRC) disease progression. Patients and methods: 210 surgical specimens comprising the ACS were immunohistochemically stained for endothelial cells (CD31), VEGF and TF. Angiogenesis quantified using Chalkley grid analysis (microvascular density; MVD), and VEGF/TF expression were semiquantitatively graded and correlated with standard prognostic indicators including 5 year follow-up. VEGF and TF were measured by ELISA in tumour specimens and normal mucosa from an additional 90 CRC patients. Results: There was a significant increase in MVD across the ACS (p < 0.0005) with significant correlations with Dukes’ stage (p  =  0.01) and lymph node involvement (p  =  0.02). The greatest increase in MVD was related to the onset of dysplasia, with an associated significant increase in VEGF expression (p < 0.0005). There was a significant relationship between VEGF and TF expression in the initial phase of the ACS (k  =  0.44, p < 0.005), although no correlation between VEGF or TF, and MVD, tumour size, Dukes’ classification, lymph node involvement or survival was found. Conclusions: These findings are the first to suggest that the angiogenic switch occurs at the onset of dysplasia in the ACS, and provide further evidence of the close association between VEGF and TF in the early stages of CRC development.

Angiogenesis, vascular endothelial growth factor and its receptors in human surgical wounds.

Angiogenesis plays an essential role in tissue repair. Vascular endothelial growth factor (VEGF) mediates angiogenesis through receptor kinases VEGF‐R1 and VEGF‐R2, and co‐receptors, neuropilins Np1 and Np2. This study examined the spatial and temporal expression of these factors in relation to angiogenesis in surgical wounds.

The role of fibrinogen and related fragments in tumour angiogenesis and metastasis

Angiogenesis, the development of new blood vessels from existing vasculature, involves the migration, proliferation and differentiation of endothelial cells and is crucial for the growth and mestastasis of tumours. A specific association between cancer and the haemostatic system has long been recognised. Haemostatic mechanisms regulate blood flow by controlling platelet adhesion and fibrin deposition, and a number of haemostatic proteins have been shown to regulate angiogenesis, either directly, by interacting with endothelial cells themselves, or indirectly, by interacting with other regulators of angiogenesis. The polypeptide fibrinogen is the central protein in the haemostasis pathway and is found deposited in the majority of human and experimental animal tumours. In this review, the evidence for the ability of fibrinogen and various protein/peptide fragment derivatives to modulate angiogenic mechanisms in vitro and to affect tumour growth and metastasis in vivo is discussed.

Tissue factor, angiogenesis and tumour progression

Tissue factor, the primary initiator of the coagulation cascade, maintains vascular integrity in response to injury. It is now recognised that, in addition to the role as a procoagulant activator, tissue factor participates in many tumour-related processes that contribute to malignant disease progression. The present review details the recent evidence supporting a role for tissue factor in tumour haemostasis, angiogenesis, metastasis and malignant cell survival. Furthermore, future research directions are discussed that may enhance our understanding of the role and regulation of this protein, which could ultimately lead to the innovative design and development of new anticancer therapies.

Transforming Growth Factor-β and Endoglin Signaling Orchestrate Wound Healing.

Physiological wound healing is a complex process requiring the temporal and spatial co-ordination of various signaling networks, biomechanical forces, and biochemical signaling pathways in both hypoxic and non-hypoxic conditions. Although a plethora of factors are required for successful physiological tissue repair, transforming growth factor beta (TGF-β) expression has been demonstrated throughout wound healing and shown to regulate many processes involved in tissue repair, including production of ECM, proteases, protease inhibitors, migration, chemotaxis, and proliferation of macrophages, fibroblasts of the granulation tissue, epithelial and capillary endothelial cells. TGF-β mediates these effects by stimulating signaling pathways through a receptor complex which contains Endoglin. Endoglin is expressed in a broad spectrum of proliferating and stem cells with elevated expression during hypoxia, and regulates important cellular functions such as proliferation and adhesion via Smad signaling. This review focuses on how the TGF-β family and Endoglin, regulate stem cell availability, and modulate cellular behavior within the wound microenvironment, includes current knowledge of the signaling pathways involved, and explores how this information may be applicable to inflammatory and/or angiogenic diseases such as fibrosis, rheumatoid arthritis and metastatic cancer.

Angiogenesis is associated with the onset of hyperplasia in human ductal breast disease.

Background:The precise timing of the angiogenic switch and the role of angiogenesis in the development of breast malignancy is currently unknown.Methods:Therefore, the expression of CD31 (pan endothelial cells (ECs)), endoglin (actively proliferating ECs), hypoxia-inducible factor-1 (HIF-1α), vascular endothelial growth factor-A (VEGF) and tissue factor (TF) were quantified in 140 surgical specimens comprising normal human breast, benign and pre-malignant hyperplastic tissue, in situ and invasive breast cancer specimens.Results:Significant increases in angiogenesis (microvessel density) were observed between normal and benign hyperplastic breast tissue (P<0.005), and between in situ and invasive carcinomas (P<0.0005). In addition, significant increases in proliferating ECs were observed in benign hyperplastic breast compared with normal breast (P<0.05) cancers and in invasive compared with in situ cancers (P<0.005). Hypoxia-inducible factor-1α, VEGF and TF expression were significantly associated with increases in both angiogenesis and proliferating ECs (P<0.05). Moreover, HIF-1α was expressed by 60–75% of the hyperplastic lesions, and a significant association was observed between VEGF and TF in ECs (P<0.005) and invasive tumour cells (P<0.01).Conclusions:These findings are the first to suggest that the angiogenic switch, associated with increases in HIF-1α, VEGF and TF expression, occurs at the onset of hyperplasia in the mammary duct, although the greatest increase in angiogenesis occurs with the development of invasion.