Angiotensinogen and Its Cleaved Derivatives Inhibit Angiogenesis

Abstract

Abstract-The members of the serine protease inhibitor (serpin) family, which share a common tertiary structure and a role as serin protease inhibitors, are involved in a variety of newly discovered functions. For example, antithrombin III exerts a strong antiangiogenic activity. Angiotensinogen, the renin substrate, has a folded structure and is a member of the noninhibitory serpin subfamily. Two other noninhibitory serpins, maspin and pigment epithelium-derived factor, have antiangiogenic properties. We investigated the antiangiogenic effect of angiotensinogen and 2 related compounds: (1) des(angiotensin I)angiotensinogen, the product of angiotensinogen cleavage by renin, and (2) the reactive center loop-cleaved angiotensinogen, which is produced after selective and limited proteolysis by the protease V8. We used well-established in vitro (endothelial cell proliferation and migration, and capillary-like tube formation on Matrigel) and in vivo (the chick chorioallantoic membrane assay) models of angiogenesis to evaluate the antiangiogenic activities of these 3 related molecules. Our data demonstrated that these compounds exerted a clear and equipotent antiangiogenic effect, thus attributing a novel function to angiotensinogen and des(angiotensin I)angiotensinogen, for which no function was previously known. , an inactive decapeptide that is converted into Ang II, the main effector of the renin-angiotensin system (RAS). Its only role known is as a substrate for renin, a highly specific aspartyl protease. Renin cleaves the N-terminal end of AGT to generate Ang I. This leaves a much larger fragment intact (97.8% of the whole amino acid sequence), called des(angiotensin I)angiotensinogen (des[Ang I]AGT), which until now did not have any known function (Figure 1 for a schematic representation of AGT and its derivatives).The biochemical, enzymological, and structural characteristics of AGT have been thoroughly investigated as it is the rate-limiting step in the first reaction of the RAS cascade: its concentration in human plasma (1 mol/L) is close to its affinity (Km) for renin. 1 The liver is the main site of AGT synthesis, but other sites include the glial cells, adipocytes, kidney, and the walls of large vessels. 2 The concentration of AGT in the plasma is regulated by several endocrine factors 3 and also depends on the genotype of the AGT gene. An AGT gene variant at position 235 (235T) is associated with a 10% to 20% increase in plasma AGT concentration and high blood pressure. 4 Des(Ang I)AGT was long considered to be a degradation product and thus has not been studied extensively. What has been suggested is that des(Ang I)AGT may inhibit the renin AGT reaction. 5,6 AGT shares amino acid sequence and structural homologies with the serine protease inhibitor (serpin) family of proteins, but it has no inhibitor activity. Indeed, like 3 other noninhibitory serpins (ovalbumin, pigment epithelial-derived factor [PEDF], and maspin 7 ), AGT does not undergo the classical stressed-relaxed pathway of the inhibitory serpin...

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