Abstract
Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.
Highlights
To ensure that refolded rhLK68 produced in E. coli retains its native conformation and maintains biological activity, an identical cDNA representing rhLK68 was expressed in Chinese hamster ovary (CHO) cells, and an endothelial cell migration assay in vitro and a chorioallantoic membrane (CAM) assay in vivo were performed
SAPK/JNK and p38 MAPK are activated in response to environmental stresses, whereas ERK1/2 are activated by growth factors and are involved in cell proliferation and differentiation
In addition to endothelial cell migration, the extracellular signal-regulated kinase (ERK) signaling pathway plays an important role in choroidal endothelial cell proliferation, only about half of the total signaling appears to be mediated by this pathway [32]
Summary
Inspired by the discovery of the potent endogenous angiogenesis inhibitor angiostatin from the urine of tumor-bearing mice [7], Schulter et al [16] reported that full size recombinant apo(a) and the naturally occurring urinary fragment of apo(a), which spans KIV-1 through KIV-4, inhibit in vitro tube formation of human microvessel endothelial cells in a fibrin matrix. Urinary apo(a) fragments showed significant inhibitory effects, they were found to be much less effective than full-size apo(a) These results suggest that other structures in apo(a), such as the C-terminal kringle domains, are primarily responsible for its anti-angiogenic activity. KV is the only apo(a) kringle domain that is homologous to plasminogen kringle 5 Based on these facts, we expressed these three kringle domains in Escherichia coli and determined the anti-angiogenic and anti-tumor activities of the recombinant protein, referred to as rhLK68. We found that rhLK68 can suppress angiogenesis-dependent tumor growth and downregulate the expression of angiogenic factors within the tumor mass
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