INTRODUCTION

In vivo the occurrence of angiogenesis is accompanied by hyperemia and vasodilation of preexisting capillaries. We have recently reported that vasoactive peptides inducing nitric oxide(NO)-mediated vasorelaxation, promote angiogenesis in vivo and endothelial cell growth and mobilization in vitro and that NO-donor drugs promote endothelial cells proliferation in vitro. We have also shown that inhibition of NO-synthase suppresses angiogenesis in vivo.

Based on these considerations we hypothesize that NO production by capillaries can be instrumental for tumor angiogenesis and could function as a transducing signal to mediate the activity of specific angiogenesis factor .

AIM

The aim of this study is to investigate the role of NO in the control of tumor angiogenesis and as a mediator of angiogenesis factor activity.

OBJECTIVES

The study will be conducted :

A) IN VITRO:

1) The ability of angiogenesis factor to affect the production of NO will be assessed in vitro on postcapillary endothelial cells isolated from coronary vessel (CVEC); cyclic GMP levels and NO synthase activity will be measured.

2) The ability of NO to regulate angiogenesis will be studied by assessing the effect of NO-generating drugs and of NO synthase inhibitors on the cellular events of angiogenesis, i.e. proliferation , migration and protease activity in CVEC.

B) IN VIVO:

The role of NO on in vivo angiogenesis will be assessed on the rabbit cornea model. The effect of NO-synthase inhibitors to affect angiogenesis will be evaluated in vivo a) on the angiogenesis process elicited by tumor cells in the rabbit cornea and b) on growth rate and vascularity of tumor xenographts.

Human tumor cell line (MCF7 and HEC) overexpressing angiogenic growth factors will be used in this project and their angiogenic activity studied in vivo in the rabbit cornea assay. In the same animal each cell line will be compared to the parent line not expressing the growth factor.

METHOD: Angiogenesis can be studied in the cornea of albino rabbits since this is an avascular and transparent tissue where growing capillaries can be easily monitored and changes quantitated by stereomicroscopic examination. This method allows the monitoring over an extended period of time of vessel growth by direct and non traumatic observation of the process. A micro pocket is surgically produced in the rabbit cornea. Angiogenesis factor activity is tested by preparing slow-release pellets incorporating the test substances into a casting solution of a ethynil-vinyl copolymer (Elvax 40, DuPont), in 10% methylene chloride (Figure 1).

Angiogenesis elicited by cell suspension can also be assessed in the rabbit cornea (Figure 2).

Observations of the implants is performed with a slit lamp stereomicroscope. Angiogenic activity is evaluated on the basis of the number and growth rate of newly formed capillaries.