Search

Nitric oxide (NO) is an important signalling molecule that acts in many tissues to regulate different physiological and pathological processes. We have contributed to demonstrate that NO stimulates angiogenesis and mediates the effect of different angiogenic molecules. In human tumors NOS expression and activity correlate with tumor growth and aggressiveness, through angiogenesis stimulation and regulation of angiogenic factor expression. Drugs affecting the NOS pathway appear promising antitumor strategies by reducing edema, inhibiting angiogenesis and facilitating the delivery of chemotherapeutical agents.

The corneal pocket assay consists of the placement of a stimulus (purified growth factor, cell suspension, tumor tissue) into a corneal pocket in order to induce a vascular outgrowth from the limbal vessels toward the implant. This assay, with respect to the other in vivo assays, such as the chorio-allantoic membrane, has the advantage of measuring only new blood vessels, since the cornea is normally avascular. By using rabbits, additional advantages are gained like the easy manipulation of the animals and monitoring of the neovascular growth and inflammatory reaction over time. Measurement of corneal angiogenesis is useful to quantify the effects of angiogenic stimuli and to evaluate the efficacy of potential inhibitors of neovascularization following local delivery or systemic treatment.

Bradykinin (BK) contributes to the inflammatory response inducing vasodilation of postcapillary venules and has been demonstrated to induce neovascular growth in subcutaneous rat sponges. In this study the ability of BK to stimulate cell growth and migration in cultured endothelium from coronary postcapillary venules (CVEC) has been investigated. BK promotes growth of endothelial cells and its mitogenic activity involves c-Fos expression. Only the Bl receptor appears to be responsible for the proliferation induced by BK and suggests that this type of receptor might be implicated in favouring angiogenesis of coronary venules. Phospholipase C (PLC) activation and the endogenous upregulation of nitric oxide synthase (NOS) were monitored in response to Bl and B2 receptor activation. BK-induced IP turnover was triggered by B2 activation, while NOS activation was linked to the Bl receptor. NOS inhibition in CVEC prevented BK/B1 induced cell growth. These results substantiate the protective and trophic role of BK on microvascular endothelium by demonstrating that Bl receptor stimulation on venular endothelium is mandatory for the angiogenic effect of BK through its coupling to NOS activation.

Angiogenesis, the formation of new microvessels from a pre-existing vascular bed, is stimulated by tissue hypoxia. Adenosine, a key extracellular signal elicited by hypoxia, plays several roles in initiation and/or modulation of angiogenesis. Adenosine governs the angiogenesis process through its actions on endothelial cell proliferation and migration. In addition, adenosine amplifies the mitogenic signal elicited by other angiogenic mediators such as nitric oxide and vascular endothelial growth factor. Through the interplay of tissue oxygen tension, adenosine, nitric oxide and vascular endothelial growth factor, a complex physiological control system ensures the matching of vascularity and metabolic needs in a variety of organs and circumstances.

Basic fibroblast growth factor (bFGF) is expressed in vascular endothelium during tumor neovascularization and angioproliferative diseases. The ultimate significance of this observation is poorly understood. We have investigated the biological consequences of endothelial cell activation by endogeneous bFGF in a mouse aortic endothelial cell line stably transfected with a retroviral expressionvector harboring a human bFGF cDNA. Selected clones expressing 24 kD, 22 kD, and/or 18 kD bFGF isoforms were characterized by a transformed morphology and an increased saturation density. bFGF transfectants showed invasive behavior and sprouting activity in three-dimensional fibrin gels and formed a complex network of branching cord-like structures connecting foci of infiltrating cells when seeded on laminin-rich basement membrane matrix (Matrigel). The invasive and morphogenetic behavior was prevented by anti-bFGF antibody, revealing the autocrine modality of the process. The biological consequences of this autocrine activation were investigated in vivo. bFGF-transfected cells gave rise to highly vascularized lesions resembling Kaposi’s sarcoma when injected in nude mice and induced angiogenesis in avascular rabbit cornea. Injected into the allantoic sac of the chick embryo they caused an increase in vascular density and formation of mangiomas in the choriallantoic membrane. In conclusion, bFGF-overexpressing endothelial cells acquire an angiogeneic phenotype and reclute quiescent endothelium originating angioproliferative lesions in vivo. These findings demonstrated that bFGF overexpression exerts an autocrine role for endothelial cells and support the notion that tumor neovascularization and angioproliferative diseases can be triggered by stimuli that induce vascular endothelium to produce its own autocrine factors(s).

In order to develop angiogenic and antiangiogenic strategies, there are concerted efforts to provide animal model for more quantitative analysis of in vivo angiogenesis. In vivo techniques consist in the rat and rabbit cornea assay, the sponge implant assay, the fibrin clots, sodium alginate beads and matrigel plugs and the chick embryo chorioallantoic membrane. In this chapter we will discuss on the avascular cornea assay and the advantages and divadvantages in the rat and the rabbits, the two animal species most used.

Screening of 84 primary human breast carcinomas for the mRNA expression of seven angiogenic polypeptides showed that the most commonly expressed and/or the most highly expressed when compared to normal breast tissue were vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor/thymidine Phosphorylase (PDECGF/TP)1. The neurokines midkine (MK) and pleiotrophin were also fairly commonly expressed in tumour but not normal tissue (unpublished data from this group and ref. 1).

Under physiological circumstances the ability of a given tissue to produce a neovascular response is under strict control, i.e. the balance between factors and events favoring or interfering with neovascular growth is in equilibrium. Modification of the normal cell/cell and cell/matrix boundaries, which maintain tubular morphology of the endothelium at the postcapillary level, is a prerequisite for capillary endothelial cell mobilization and proliferation to constitute a neovascular response.