The presence of immunoreactive fragments of apolipoprotein(a), apo(a), have been shown in the plasma, urine anc atheromas of human subjects. Moreover, derivatives of lipoprotein(a), Lp(a), and apo(a) can be generated in vitrc by the action of enzymes of the elastase and metalloproteinase (MMP) families. The goal of our proposed studies is to shed light on these products, in terms of their potential participation in the cardiovascular pathogenicity ol Lp(a). In particular, we wish to test the hypothesis that enzymes of the MMP family shown in vitro to cleave Lp(a; in the hot spot of the linker region between kringle IV-4 and IV-5 of apo(a), generate at tissue sites, two mair derivatives. One, miniLp(a), a particle that has as a protein moiety apoB 100 linked to P2, the C-terminal fragmen of apo(a) able to bind to members of the vascular matrix. The other, F1 representing the N- terminal domain of spo(a) containing the kringle IV-2 repeats and, lacking matrix binding function. A portion of F1 once releasec from apo(a) at tissue sites, would return to the plasma and then rapidly excreted into the urine. In turn. F2. eithe free or a member of a miniLp(a), would be preferentially retained at the sites of formation, preferentially in the sub endotheial intima where it undergoes atherogenic changes. This hypothesis wifi be tested by four relatec approaches: 1) structural, functional and immunological studies on the properties of apo(a) in order to define its various domains, and the basis for the cleavage specificities by MMPs; 2) on the premise that the linker 4 is thc one most susceptible to MMP cleavage, express in vitro apo(a) species having linker 4 mutated to be resistant t MMP cleavage, and then compare the in vitro and in vivo properties of these mutants with those of their wild-type counterpart; 3) introduce via adenovirus technology in apoE mice and crosses between apoE-/- and human apoB100 transgenics in different stages of atherogenesis, either Fl, P2, wild-type or linker 4-mutated apo(a) anc assess the localization of these products in unaffected and lesion areas along with measurements of MMI activities; 4) study surgical segments of human carotid arteries with either stable or unstable plaques to determine using immunochemical and chemical methods, the comparative localization of apo(a) and fragments in these tw types of plaques and also determine whether the fragments resemble those generated in vitro from the digestion o Lp(a)/apo(a) with MMPs. The combined in vitro and in vivo studies are expected to improve our knowledge oi the properties of the various domains of apo(a) and shed light on the potential role that MMP-mediated proteoIysis may play in the atherogenicity of Lp(a)/apo(a) in the context of the general inflammatory theory of atherosclerosis.
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