Amiloride inhibition of angiogenesis in vitro.

Angiogenesis is important to such processes as normal embryonic development and tissue growth, and is also a central feature of diseases such as diabetic retinopathy and the growth of solid tumors. Understanding the basic events governing angiogenesis has therefore attracted great interest. The ion channel blocking agent, amiloride, has been shown to inhibit angiogenesis in an in vivo model (Lansing et al., '91). This suggested a vital role for Na(+)-coupled transport processes in angiogenesis. A large number of structural analogues of amiloride have been synthesized (Kleyman and Cragoe, '88), and many of these are well characterized with respect to biological activity. These analogues present an opportunity to dissect the process of angiogenesis and identify potentially important physiological events. In this report we describe the effects of amiloride on an in vitro model for angiogenesis employing vascularized tissue explants. Amiloride inhibits capillary morphogenesis completely and reversibly at concentrations as low as 134 microM. It appears to act by blocking endothelial cell proliferation, but not migration. Inhibition is heightened by the introduction of hydrophobic groups on the terminal guanidino nitrogen atom, or on the 5-amino position. An analogue substituted at both of these positions is 30-fold more potent than the parent compound. Of amiloride's known biological activities, these results most closely correlate with the inhibition of Ca2+ transport processes, and thereby suggest an important role for Ca2+ transport in capillary morphogenesis.