Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier.

Effective delivery of therapeutics to the brain is challenging. Molecular shuttles use receptors expressed on brain endothelial cells to deliver therapeutics. Antibodies targeting transferrin receptor (TfR) have been widely developed as molecular shuttles. However, the TfR-based approach raises concerns about safety and developmental burden. Here, we report insulin-like growth factor 1 receptor (IGF1R) as an ideal target for the molecular shuttle. We also describe Grabody B, an antibody against IGF1R, as a molecular shuttle. Grabody B has broad cross-species reactivity and does not interfere with IGF1R-mediated signaling. We demonstrate that administration of Grabody B-fused anti-alpha-synuclein (α-Syn) antibody induces better improvement in neuropathology and behavior in a Parkinson's disease animal model than the therapeutic antibody alone due to its superior serum pharmacokinetics and enhanced brain exposure. The results indicate that IGF1R is an ideal shuttle target and Grabody B is a safe and efficient molecular shuttle.

Conformation-specific Antibodies Targeting Aggregated Forms of α-synuclein Block the Propagation of Synucleinopathy.

Abnormal aggregation of α-synuclein is a key element in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. α-synuclein aggregation spreads through various brain regions during the course of disease progression, a propagation that is thought to be mediated by the secretion and subsequent uptake of extracellular α-synuclein aggregates between neuronal cells. Thus, aggregated forms of this protein have emerged as promising targets for disease-modifying therapy for PD and related diseases. Here, we generated and characterized conformation-specific antibodies that preferentially recognize aggregated forms of α-synuclein. These antibodies promoted phagocytosis of extracellular α-synuclein aggregates by microglial cells and interfered with cell-to-cell propagation of α-synuclein. In an α-synuclein transgenic model, passive immunization with aggregate-specific antibodies significantly ameliorated pathological phenotypes, reducing α-synuclein aggregation, gliosis, inflammation, and neuronal loss. These results suggest that conformation-specific antibodies targeting α-synuclein aggregates are promising therapeutic agents for PD and related synucleinopathies.

Synergistic antitumor activity of a DLL4/VEGF bispecific therapeutic antibody in combination with irinotecan in gastric cancer.

Notch signaling has been identified as a critical pathway in gastric cancer (GC) progression and metastasis, and inhibition of Delta-like ligand 4 (DLL4), a Notch ligand, is suggested as a potent therapeutic approach for GC. Expression of both DLL4 and vascular endothelial growth factor receptor 2 (VEGFR2) was similar in the malignant tissues of GC patients. We focused on vascular endothelial growth factor (VEGF), a known angiogenesis regulator and activator of DLL4. Here, we used ABL001, a DLL4/VEGF bispecific therapeutic antibody, and investigated its therapeutic effect in GC. Treatment with human DLL4 therapeutic antibody (anti-hDLL4) or ABL001 slightly reduced GC cell growth in monolayer culture; however, they significantly inhibited cell growth in 3D-culture, suggesting a reduction in the cancer stem cell population. Treatment with anti-hDLL4 or ABL001 also decreased GC cell migration and invasion. Moreover, the combined treatment of irinotecan with anti-hDLL4 or ABL001 showed synergistic antitumor activity. Both combination treatments further reduced cell growth in 3D-culture as well as cell invasion. Interestingly, the combination treatment of ABL001 with irinotecan synergistically reduced the GC burden in both xenograft and orthotopic mouse models. Collectively, DLL4 inhibition significantly decreased cell motility and stem-like phenotype and the combination treatment of DLL4/VEGF bispecific therapeutic antibody with irinotecan synergistically reduced the GC burden in mouse models. Our data suggest that ABL001 potentially represents a potent agent in GC therapy. Further biochemical and pre-clinical studies are needed for its application in the clinic. [BMB Reports 2020; 53(10): 533-538].

ABL001, a Bispecific Antibody Targeting VEGF and DLL4, with Chemotherapy, Synergistically Inhibits Tumor Progression in Xenograft Models.

Delta-like-ligand 4 (DLL4) is a promising target to augment the effects of VEGF inhibitors. A simultaneous blockade of VEGF/VEGFR and DLL4/Notch signaling pathways leads to more potent anti-cancer effects by synergistic anti-angiogenic mechanisms in xenograft models. A bispecific antibody targeting VEGF and DLL4 (ABL001/NOV1501/TR009) demonstrates more potent in vitro and in vivo biological activity compared to VEGF or DLL4 targeting monoclonal antibodies alone and is currently being evaluated in a phase 1 clinical study of heavy chemotherapy or targeted therapy pre-treated cancer patients (ClinicalTrials.gov Identifier: NCT03292783). However, the effects of a combination of ABL001 and chemotherapy on tumor vessels and tumors are not known. Hence, the effects of ABL001, with or without paclitaxel and irinotecan were evaluated in human gastric or colon cancer xenograft models. The combination treatment synergistically inhibited tumor progression compared to each monotherapy. More tumor vessel regression and apoptotic tumor cell induction were observed in tumors treated with the combination therapy, which might be due to tumor vessel normalization. Overall, these findings suggest that the combination therapy of ABL001 with paclitaxel or irinotecan would be a better clinical strategy for the treatment of cancer patients.

High delta-like ligand 4 expression correlates with a poor clinical outcome in gastric cancer.

Background: Emerging evidence suggests that delta-like ligand 4 (DLL4) and other members of the Notch pathway may offer new targets for development of anti-angiogenesis drugs for the treatment of several tumor types. However, the role of DLL4 in gastric cancer (GC) remains unclear. In this study, we investigated the impact of DLL4 overexpression on recurrence and survival in gastric cancer (GC) patients. Methods: DLL4 expression levels were evaluated by immunohistochemistry in tissue samples from 336 GC patients. Samples were classified into high and low DLL4 expression according to a cut-off of 50% positively stained cells. The correlation between DLL4 expression and clinicopathological parameters, disease-free survival (DFS), and overall survival (OS) were statistically analyzed. Results: High DLL4 expression was observed in 67 (19.9%) of the 336 GC patients. After a median follow-up duration of 54.97 months [95% confidence interval (CI), 52.40-57.55 months), patients at stage II-IV with high DLL4 expression showed significantly poorer DFS compared with those at the same stage but with low DLL4 expression [not reached (NR) for both cohorts, hazard ratio (HR) 0.73 (95% CI, 0.38-1.40); p = 0.007]. Likewise, GC patients with high DLL4 expression had a significantly shorter OS following curative surgery compared to those with low DLL4 expression [NR for both groups, HR 0.56 (95% CI, 0.32-0.96; p = 0.002]. High DLL4 expression had a greater influence on DFS in stage IIIb/IV patients than in patients at early stages [34.87 vs. 10.1 months; HR, 0.44 (95% CI, 0.19-0.96); p = 0.004]. Moreover, stage IIIb/IV patients with high DLL4 expression had a significantly shorter OS after surgery than those with low DLL4 expression [58.87 vs. 16.93 months, HR 0.39 (95% CI, 0.16-0.99), p = 0.001). Conclusion: High DLL4 expression was observed in 19.9% of GC patients and was significantly associated with poor survival following curative surgery. Given its prevalence in the GC cohort with a poor prognosis, DLL4 is a potential therapeutic target.

Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis.

Several angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathway have been approved for cancer treatment. However, VEGF inhibitors alone were shown to promote tumor invasion and metastasis by increasing intratumoral hypoxia in some preclinical and clinical studies. Emerging reports suggest that Delta-like ligand 4 (Dll4) is a promising target of angiogenesis inhibition to augment the effects of VEGF inhibitors. To evaluate the effects of simultaneous blockade against VEGF and Dll4, we developed a bispecific antibody, HD105, targeting VEGF and Dll4. The HD105 bispecific antibody, which is composed of an anti-VEGF antibody (bevacizumab-similar) backbone C-terminally linked with a Dll4-targeting single-chain variable fragment, showed potent binding affinities against VEGF (KD: 1.3 nM) and Dll4 (KD: 30 nM). In addition, the HD105 bispecific antibody competitively inhibited the binding of ligands to their receptors, i.e., VEGF to VEGFR2 (EC50: 2.84 ± 0.41 nM) and Dll4 to Notch1 (EC50: 1.14 ± 0.06 nM). Using in vitro cell-based assays, we found that HD105 effectively blocked both the VEGF/VEGFR2 and Dll4/Notch1 signaling pathways in endothelial cells, resulting in a conspicuous inhibition of endothelial cell proliferation and sprouting. HD105 also suppressed Dll4-induced Notch1-dependent activation of the luciferase gene. In vivo xenograft studies demonstrated that HD105 more efficiently inhibited the tumor progression of human A549 lung and SCH gastric cancers than an anti-VEGF antibody or anti-Dll4 antibody alone. In conclusion, HD105 may be a novel therapeutic bispecific antibody for cancer treatment.

Development of antibody-based c-Met inhibitors for targeted cancer therapy.

Signaling pathways mediated by receptor tyrosine kinases (RTKs) and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics.

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.

Adverse functional effects of chemotherapy on whole-brain metabolism: a PET/CT quantitative analysis of FDG metabolic pattern of the "chemo-brain".

PURPOSE: With its unique ability to assess function through metabolism, FDG PET may provide key insight into the adverse effects of chemotherapy on the brain by shedding light on its cognitive and psychological consequences, a well-known phenomenon in cancer patients who undergo such treatments. PATIENTS AND METHODS: Twenty-one patients with untreated non-Hodgkin lymphoma and no involvement of the central nervous system were selected from an existing database for this investigation. An adaptive threshold imaging software called ROVER was used to analyze and quantify FDG PET scans for assessing whole-brain metabolism. A 3D spherical region of interest was drawn surrounding the cerebral cortex, excluding subcortical and non-cortical areas. RESULTS: We noted a mean reduction of 1308.86 ± 394.74 SUV-cc (P = 0.03) in the whole-brain cortical glycolysis after standard chemotherapy, which translated to a decrease of 16.9 ± 5.04% (P = 0.03) in the measured structures. CONCLUSION: These results demonstrate the potential role of such adaptive quantitative techniques in studying underlying biological effects of chemotherapy and offer a promising avenue for future research in this field.

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.

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.

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.

Human apolipoprotein(a) kringle V inhibits ischemia-induced retinal neovascularization via suppression of fibronectin-mediated angiogenesis.

Retinal neovascularization is observed in progression of diabetic retinopathy. New vessels grow into the vitreous cavity in proliferative diabetic retinopathy, resulting in traction retinal detachment and vitreous hemorrhage. To overcome the catastrophic visual loss due to these complications, efforts have been focused on the treatment of retinal neovascularization. In this study, we demonstrated the inhibitory effect of recombinant human apolipoprotein(a) kringle V (rhLK8) in an animal model of ischemia-induced retinal neovascularization. rhLK8 induced no definite toxicity on endothelial cells and retinal tissues at the therapeutic dosage. Interestingly, rhLK8 showed antiangiogenic effect, particularly on fibronectin-mediated migration of endothelial cells. Further experiments demonstrated high binding affinity of rhLK8 to α3ß1 integrin, and suppression of it might be the mechanism of antiangiogenic effect of rhLK8. Furthermore, rhLK8 inhibited phosphorylation of focal adhesion kinase, resulting in suppression of activation of consequent p130CAS-Jun NH(2)-terminal kinase. Taken together, our data suggested the possible application of rhLK8 in the treatment of retinal neovascularization by suppression of fibronectin-mediated angiogenesis.

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.

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 monoclonal antibody against the paraneoplastic pemphigus (PNP) antigen, envoplakin: cDNA sequences encoding the variable regions of heavy and light chains.

Paraneoplastic pemphigus (PNP) is an acquired autoimmune disorder characterized by severe mucosal erosion, and polymorphous cutaneous lesions associated with neoplasia. PNP patients have circulating autoantibodies that bind to stratified and nonstratified epithelia. Previously, we showed that envoplakin was a component of the antigen complex recognized by PNP sera. In the present study we generated a monoclonal antibody, EVP-YS, against human envoplakin. The antibody bound to keratinocyte cell surfaces and reacted with the 210-kDa PNP antigen, confirming its specificity for envoplakin. The variable regions of the heavy (H) and light (L) chain genes were cloned from the hybridoma and shown to belong to mouse H chain subgroup III and kappa light chain subgroup V, respectively. The L chain of EVP-YS was 98% identical to the k chains of some autoantibodies and anti-nucleic acid antibodies, and had an identical amino acid sequence in all three complementary determining regions, suggesting that the H chains determine the specificity of the EVP-YS-envoplakin interaction. The EVP-YS antibody can be used to evaluate the sensitivity and specificity of clinical, histological, and immunological criteria for diagnosing PNP.

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.

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.

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.

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.