Antiangiogenic kringles derived from human plasminogen and apolipoprotein(a) inhibit fibrinolysis through a mechanism that requires a functional lysine-binding site.

Many proteins in the fibrinolysis pathway contain antiangiogenic kringle domains. Owing to the high degree of homology between kringle domains, there has been a safety concern that antiangiogenic kringles could interact with common kringle proteins during fibrinolysis leading to adverse effects in vivo. To address this issue, we investigated the effects of several antiangiogenic kringle proteins including angiostatin, apolipoprotein(a) kringles IV(9)-IV(10)-V (LK68), apolipoprotein(a) kringle V (rhLK8) and a derivative of rhLK8 mutated to produce a functional lysine-binding site (Lys-rhLK8) on the entire fibrinolytic process in vitro and analyzed the role of lysine binding. Angiostatin, LK68 and Lys-rhLK8 increased clot lysis time in a dose-dependent manner, inhibited tissue-type plasminogen activator-mediated plasminogen activation on a thrombin-modified fibrinogen (TMF) surface, showed binding to TMF and significantly decreased the amount of plasminogen bound to TMF. The inhibition of fibrinolysis by these proteins appears to be dependent on their functional lysine-binding sites. However, rhLK8 had no effect on these processes owing to an inability to bind lysine. Collectively, these results indicate that antiangiogenic kringles without lysine binding sites might be safer with respect to physiological fibrinolysis than lysine-binding antiangiogenic kringles. However, the clinical significance of these findings will require further validation in vivo.

Suppression of colorectal cancer liver metastasis and extension of survival by expression of apolipoprotein(a) kringles.

The formation of hepatic metastases in colorectal cancer is the main cause of patient death. Current therapies directed at hepatic metastasis of colorectal cancer have had minimal impact on outcome. Therefore, alternative treatment strategies for liver metastasis require development. The present study was performed to evaluate the application of cDNA of LK68 encoding apolipoprotein(a) kringles IV-9, IV-10, and V as possible candidates for gene therapy treatment of this life-threatening disease. The murine colorectal cancer cell line CT26 was transduced ex vivo with LK68 cDNA via retroviral gene transfer, and an experimental model of hepatic metastasis was established by injecting LK68-expressing and control cells into the spleens of BALB/c mice. Expression of LK68 did not affect the growth characteristics and viability of transduced CT26 cells in vitro. LK68 produced from CT26 cells substantially inhibited the migration of endothelial cells in vitro. In vivo, substantial suppression of liver metastasis and prolonged survival were observed in mice bearing LK68-expressing CT26 cells, compared with controls. LK68-expressing liver metastases were restricted to smaller sizes and displayed decreased microvessel density and increased tumor cell apoptosis. Our data collectively indicate that LK68 suppresses angiogenesis-dependent progression of prevascular micrometastases to macroscopic tumors and their growth, which are clinically accessible and biologically relevant therapeutic targets. We propose that antiangiogenic gene therapy with LK68 is a promising strategy for the treatment of colorectal cancer liver metastasis.

Suppression of colorectal cancer liver metastasis by apolipoprotein(a) kringle V in a nude mouse model through the induction of apoptosis in tumor-associated endothelial cells.

The formation of liver metastases in colorectal cancer patients is the primary cause of patient death. Current therapies directed at liver metastasis from colorectal cancer have had minimal impact on patient outcomes. Therefore, the development of alternative treatment strategies for liver metastasis is needed. In the present study, we demonstrated that recombinant human apolipoprotein(a) kringle V, also known as rhLK8, induced the apoptotic turnover of endothelial cells in vitro through the mitochondrial apoptosis pathway. The interaction of rhLK8 with glucose-regulated protein 78 (GRP78) may be involved in the induction of apoptosis because the inhibition of GRP78 by GRP78-specific antibodies or siRNA knockdown inhibited the rhLK8-mediated apoptosis of human umbilical vein endothelial cells in vitro. Next, to evaluate the effects of rhLK8 on angiogenesis and metastasis, an experimental model of liver metastasis was established by injecting a human colorectal cancer cell line, LS174T, into the spleens of BALB/c nude mice. The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls. The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone. Histological observation showed significant induction of apoptosis among tumor-associated endothelial cells in liver metastases from rhLK8-treated mice compared with control mice. Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases.

Antiangiogenic Therapy with Human Apolipoprotein(a) Kringle V and Paclitaxel in a Human Ovarian Cancer Mouse Model.

INTRODUCTION: The present study compared the effect of combination therapy using human apolipoprotein(a) kringle V (rhLK8) to conventional chemotherapy with paclitaxel for human ovarian carcinoma producing high or low levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Human ovarian carcinoma cells producing high (SKOV3ip1) or low (HeyA8) levels of VEGF were implanted into the peritoneal cavity of female nude mice. Seven days later, mice were randomized into four groups: control (vehicle), paclitaxel [5 mg/kg, weekly intraperitoneal (i.p.) injection], rhLK8 (50 mg/kg, daily i.p. injection), or the combination of paclitaxel and rhLK8. Mice were treated for 4 weeks and examined by necropsy. RESULTS: In mice implanted with SKOV3ip1 cells, rhLK8 treatment had no significant effect on tumor incidence or the volume of ascites but induced a significant decrease in tumor weight compared with control mice. Paclitaxel significantly reduced tumor weight and ascites volume, and combination treatment with paclitaxel and rhLK8 had an additive therapeutic effect. Similarly, in HeyA8 mice, the effect of combination treatment on tumor weight and tumor incidence was statistically significantly greater than that of paclitaxel or rhLK8 alone. Immunohistochemical analysis showed a significant decrease in microvessel density and a marked increase of apoptosis in tumor and tumor-associated endothelial cells in response to combination treatment with paclitaxel and rhLK8. CONCLUSION: Collectively, these results suggest that antiangiogenic therapy with rhLK8 in combination with taxane-based conventional chemotherapy could be effective for the treatment of ovarian carcinomas, regardless of VEGF status.

Immunoglobulin Fc domain fusion to apolipoprotein(a) kringle V significantly prolongs plasma half-life without affecting its anti-angiogenic activity.

Angiogenesis is crucial for tumor growth and metastasis. Blocking this process is, therefore, a potentially powerful approach for the treatment of cancer. Human apolipoprotein(a) kringle V (rhLK8) is an angiogenesis inhibitor and is currently under development as an anti-cancer therapeutic. However, a relatively short in vivo half-life limits its widespread clinical use. This study was performed to evaluate whether fusion of an Fc domain to rhLK8 can extend plasma half-life. RhLK8-Fc fusion protein was expressed in CHO DG44 cells as a dimer and was readily purified by protein G affinity chromatography. The anti-angiogenic activity of rhLK8-Fc was similar to that of rhLK8, as determined by migration and tube formation assays with endothelial cells in vitro and a chorioallantoic membrane assay in vivo. Pharmacokinetic profiles in mice after single intravenous administration of rhLK8 or rhLK8-Fc showed that Fc fusion significantly increased the elimination half-life (t(½)) and the systemic exposure (AUC(inf)) of the protein, in parallel with a significant decrease in total clearance (CL). These data suggest that Fc fusion to rhLK8 is a powerful strategy for extending the plasma half-life of rhLK8 without affecting its anti-angiogenic activity, and could thus improve the clinical applicability of rhLK8.

Characterization of CD45-/CD31+/CD105+ circulating cells in the peripheral blood of patients with gynecologic malignancies.

PURPOSE: Circulating endothelial cells (CEC) have been widely used as a prognostic biomarker and regarded as a promising strategy for monitoring the response to treatment in several cancers. However, the presence and biologic roles of CECs have remained controversial for decades because technical standards for the identification and quantification of CECs have not been established. Here, we hypothesized that CECs detected by flow cytometry might be monocytes rather than endothelial cells. EXPERIMENTAL DESIGN: The frequency of representative CEC subsets (i.e., CD45(-)/CD31(+), CD45(-)/CD31(+)/CD146(+), CD45(-)/CD31(+)/CD105(+)) was analyzed in the peripheral blood of patients with gynecologic cancer (n = 56) and healthy volunteers (n = 44). CD45(-)/CD31(+) cells, which are components of CECs, were isolated and the expression of various markers (CD146, CD105, vWF, and CD144 for endothelial cells; CD68 and CD14 for monocytes) was examined by immunocytochemistry. RESULTS: CD45(-)/CD31(+)/CD105(+) cells were significantly increased in the peripheral blood of patients with cancer, whereas evaluation of CD45(-)/CD31(+)/CD146(+) cells was not possible both in patients with cancer and healthy controls due to the limited resolution of the flow cytometry. Immunocytochemistry analyses showed that these CD45(-)/CD31(+)/CD105(+) cells did not express vWF and CD146 but rather CD144. Furthermore, CD45(-)/CD31(+)/CD105(+) cells uniformly expressed the monocyte-specific markers CD14 and CD68. These results suggest that CD45(-)/CD31(+)/CD105(+) cells carry the characteristics of monocytes rather than endothelial cells. CONCLUSIONS: Our data indicate that CD45(-)/CD31(+)/CD105(+) circulating cells, which are significantly increased in the peripheral blood of patients with gynecologic cancer, are monocytes rather than endothelial cells. Further investigation is required to determine the biologic significance of their presence and function in relation with angiogenesis.

Repeated intravenous infusion of human apolipoprotein(a) kringle V is associated with reversible dose-dependent acute tubulointerstitial nephritis without affecting glomerular filtration function.

Because anti-angiogenic agents have shown various toxicities in clinical applications, the determination of their toxicities and their reversibility is important in the design of clinical trials. This study was performed to investigate the potential toxicities of an angiogenesis inhibitor, apolipoprotein(a) (Apo(a)) kringle V (rhLK8) in rats. Rats administered an intravenous (IV) bolus injection of rhLK8 (200 mg/kg) for 7 days showed significant increases in serum blood urea nitrogen (BUN), creatinine and the BUN/creatinine ratio, which was compatible with acute tubulointerstitial nephritis (TIN) in pathological examination. Because anti-angiogenic therapies are usually based on long-term treatment strategies, rats were administered 200 mg/kg/day of rhLK8 by intravenous infusion for 28 days. Rats receiving 200 mg/kg of rhLK8 showed abnormal serological and histologic findings, but their levels returned to within normal ranges 2 weeks after the cessation of administration. The creatinine clearance rate (CCr) was not affected by rhLK8 treatment. Collectively, our data indicate that the intravenous infusion of rhLK8 at therapeutic doses may induce renal toxicities, such as acute TIN, but these toxicities are clinically tolerable and reversible with close monitoring and a recovery period.

Targeted antivascular therapy with the apolipoprotein(a) kringle V, rhLK8, inhibits the growth and metastasis of human prostate cancer in an orthotopic nude mouse model.

Antivascular therapy has emerged as a rational strategy to improve the treatment of androgen-independent prostate cancer owing to the necessity of establishing a vascular network for the growth and progression of the primary and metastatic tumor. We determined whether recombinant human apolipoprotein(a) kringle V, rhLK8, produces therapeutic efficacy in an orthotopic human prostate cancer animal model. Fifty thousand androgen-independent human prostate cancer cells (PC-3MM2) were injected into the prostate of nude mice. After 3 days, these mice were randomized to receive the vehicle solution (intraperitoneally [i.p.], daily), paclitaxel (8 mg/kg i.p., weekly), rhLK8 (50 mg/kg i.p., daily), or a combination of paclitaxel and rhLK8 for 4 weeks. Treatment with paclitaxel or rhLK8 alone did not show significant therapeutic effects on tumor incidence or on tumor size compared with the control group. The combination of rhLK8 and paclitaxel significantly reduced tumor size and incidence of lymph node metastasis. Significant reduction in microvessel density and cellular proliferation and induction of apoptosis of tumor cells, and tumor-associated endothelial cells, were also achieved. Similarly, PC-3MM2 tumors growing in the tibia showed significant suppression of tumor growth and lymph node metastasis by the combination treatment with rhLK8 and paclitaxel. The integrity of the bone was significantly preserved, and apoptosis of tumor cells and tumor-associated endothelial cells was increased. In conclusion, these results suggest that targeting the tumor microenvironment with the antivascular effect of rhLK8 combined with conventional cytotoxic chemotherapy could be a new and effective approach in the treatment of androgen-independent prostate cancer and their metastases.

Expression of human apolipoprotein(a) kringles in colon cancer cells suppresses angiogenesis-dependent tumor growth and peritoneal dissemination.

BACKGROUND: Anti-angiogenesis therapy has been regarded as a promising treatment of cancer based on the fact that most tumors and their metastasis are angiogenesis-dependent. Gene therapy can potentially expand the horizons of tumor angiogenesis therapy by virtue of its ability to produce high concentrations of therapeutic agents in a local area for a sustained period. The present study was performed to evaluate the therapeutic potential of gene therapy for the treatment of cancer and metastasis. METHODS: The murine colon carcinoma cell line CT26 was manipulated ex vivo to express an anti-angiogenic molecule, LK68, consisting of human apolipoprotein(a) kringle domains, KIV(9)-KIV(10)-KV, using retrovirus-mediated gene transfer. Its effects on colon tumor growth and metastasis were evaluated in experimental animal models established by injecting LK68-expressing and control CT26 cells subcutaneously or into the peritoneal cavity of BALB/c mice, respectively. RESULTS: Expression of LK68 significantly suppressed colon tumor growth in mice, but did not influence the growth of tumor cells in vitro. Immunohistochemical analysis of tumor tissues revealed a significant reduction in microvessel density in LK68-expressing tumors. Thus, the suppression of tumor growth appears to result mainly from inhibition of tumor angiogenesis. This decrease in vessel density is correlated with a notable increase in tumor cell apoptosis in vivo, but has no influence on proliferation. Moreover, expression of LK68 prevents peritoneal dissemination, and consequently improves overall host survival. CONCLUSIONS: These results collectively indicate that a gene therapy strategy using LK68 cDNA is useful for the treatment for both colon tumor growth and peritoneal dissemination.

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.

Most proangiogenic factors exert their biological effects primarily by activating extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/Akt signaling pathways. These pathways appear to play a critical role in endothelial cell migration, because selective inhibition of either ERK or PI3-K/Akt signaling almost completely prevented endothelial cell migration. Recently, we demonstrated that a truncated kringle domain of human apolipoprotein(a), termed rhLK68, inhibits endothelial cell migration in vitro. However, its mechanism of action was not well defined. In this study, we determined the effects of rhLK68 on ERK1/2 and PI3-K/Akt signaling pathways to explore the molecular mechanism of rhLK68-mediated inhibition of endothelial cell migration. Treatment with rhLK68 inhibited ERK1/2 phosphorylation but did not influence Akt activation. Interestingly, an inhibitor of protein-tyrosine phosphatase, sodium orthovanadate, dose-dependently reversed both rhLK68-induced dephosphorylation of ERK1/2 and decreased migration of endothelial cells, whereas rhLK68 showed no significant effects on MEKs phosphorylation. In conclusion, these results indicate that inhibition of endothelial cell migration by rhLK68 may be achieved by interfering with ERK1/2 activation via a protein-tyrosine phosphatase-dependent pathway.

Inhibition of angiogenesis and angiogenesis-dependent tumor growth by the cryptic kringle fragments of human apolipoprotein(a).

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.

Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases.

Apolipoprotein(a) [apo(a)] contains the largest numbers of kringle domains identified to date. Of these, apo(a) kringle V shows significant sequence homology with plasminogen kringle 5, which is reported to be a potent angiogenesis inhibitor. To determine the effects of apo(a) kringle V on angiogenesis, it was expressed as a soluble protein (termed rhLK8) in Pichia pastoris and its in vitro and in vivo anti-angiogenic properties were examined. rhLK8 inhibited the migration of human umbilical vein endothelial cells in vitro in a dose-dependent manner. This function was associated with the down-regulation of the activation of focal adhesion kinase and the inhibition of the consequent formation of actin stress fibers/focal adhesions. rhLK8 also inhibited new capillary formation in vivo, as assessed by the chick chorioallantoic membrane assay and the Matrigel plug assay. These results indicate that rhLK8 may be an effective angiogenesis inhibitor both in vitro and in vivo.