Inhibition of Angiogenesis and Angiogenesis-dependent Tumor Growth by the Cryptic Kringle Fragments of Human Apolipoprotein(a)

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 antiangiogenic and anti-tumor molecule.

A Truncated Kringle Domain of Human Apolipoprotein(a) Inhibits the Activation of Extracellular Signal-regulated Kinase 1 and 2 through a Tyrosine Phosphatase-dependent Pathway

“…Wound Migration Assay-The effect of rhLK68 on HUVEC migration stimulated by angiogenic factors including bFGF, VEGF, and HGF was assayed in a monolayer denudation assay as previously described (10). Confluent HUVECs were wounded by scraping, washed twice with phosphate-buffered saline to remove cellular debris, and incubated in endothelial cell basal medium-2 supplemented with 1% fetal bovine serum for 24 h, following which rhLK68 proteins (1 or 3 M) were added.…”

“…Preparation of Recombinant Human LK68 -Recombinant human LK68 (rhLK68), which comprises cryptic kringle fragments of apo(a) containing KIV-9, KIV-10, and KV, was expressed and purified from Escherichia coli as described previously (10). Briefly, rhLK68 expressed as an inclusion body was collected, refolded in vitro, and purified using lysine-Sepharose column chromatography.…”

“…Although the physiological and biochemical roles of apo(a) kringle domains remain to be elucidated, there exists some evidence suggesting that they can inhibit angiogenesis in vitro (8) and suppress tumor growth in vivo (9). Recently, we have demonstrated that a recombinant peptide comprising the apo(a) kringle domains (rhLK68) that contains KIV-9, KIV-10, and KV inhibits bFGF-stimulated endothelial cell mitogenesis and migration in vitro and suppresses the growth of human lung and colon tumors in nude mice (10). However, its mechanism of action was not clearly elucidated.…”

Evidence-based assessment of lipoprotein(a) as a risk biomarker for cardiovascular diseases – Some answers and still many questions

“…Additional assays for Lp (a) oxidation are needed 101 , as are more studies to address the implication of Lp (a) Some questions remain in relation to the controversial anti-tumor function of Lp(a) 110 . In experimental studies a reduction of tumor cells growth by apo(a) was demonstrated 111,112 . The degradation of apo(a) inside of Lp(a), produces various size of kringles and this process was associated with anti-angiogenesis and anti-tumor properties 110 .…”

Adeno-associated virus-mediated expression of apolipoprotein (a) kringles suppresses hepatocellular carcinoma growth in mice

“…8 Earlier studies showed an anti-angiogenic activity of apo(a), suggesting a possible role for apo(a) in bridging atherogenesis and angiogenesis in vivo. 8,9 Recently, Kim et al 10 have demonstrated that apo(a) kringle LK68, consisting of KIV-9-KIV-10-KV, inhibits the proliferation and migration of endothelial cells in vitro and suppresses angiogenesis-dependent growth of human colon (HCT-15) and lung (A549) tumors in vivo when administered systemically. LK68-expressing murine colorectal cancer cells (CT26) restricted hepatic metastases to smaller sizes, prevented peritoneal dissemination, and significantly suppressed colon tumor growth in different experimental models.…”