Serum anti-AP3D1 antibodies are risk factors for acute ischemic stroke related with atherosclerosis.

Atherosclerosis has been considered as the main cause of morbidity, mortality, and disability worldwide. The first screening for antigen markers was conducted using the serological identification of antigens by recombinant cDNA expression cloning, which has identified adaptor-related protein complex 3 subunit delta 1 (AP3D1) as an antigen recognized by serum IgG antibodies of patients with atherosclerosis. Serum antibody levels were examined using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using a recombinant protein as an antigen. It was determined that the serum antibody levels against AP3D1 were higher in patients with acute ischemic stroke (AIS), transient ischemic attack, diabetes mellitus (DM), cardiovascular disease, chronic kidney disease (CKD), esophageal squamous cell carcinoma (ESCC), and colorectal carcinoma than those in the healthy donors. The area under the curve values of DM, nephrosclerosis type of CKD, and ESCC calculated using receiver operating characteristic curve analysis were higher than those of other diseases. Correlation analysis showed that the anti-AP3D1 antibody levels were highly associated with maximum intima-media thickness, which indicates that this marker reflected the development of atherosclerosis. The results of the Japan Public Health Center-based Prospective Study indicated that this antibody marker is deemed useful as risk factors for AIS.

Association of serum levels of antibodies against MMP1, CBX1, and CBX5 with transient ischemic attack and cerebral infarction.

Transient ischemic attack (TIA) is a predictor for cerebral infarction (CI), and early diagnosis of TIA is extremely important for the prevention of CI. We set out to identify novel antibody biomarkers for TIA and CI, and detected matrix metalloproteinase 1 (MMP1), chromobox homolog 1 (CBX1), and chromobox homolog 5 (CBX5) as candidate antigens using serological identification of antigens by recombinant cDNA expression cloning (SEREX) and Western blotting to confirm the presence of serum antibodies against the antigens. Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) revealed that serum antibody levels were significantly higher in patients with TIA or acute-phase CI (aCI) compared with healthy donors (P < 0.01). Spearman's correlation analysis and multivariate logistic regression analysis demonstrated that levels of anti-MMP1, anti-CBX1, and anti-CBX5 antibodies were associated with age, cigarette-smoking habits, and blood pressure. Thus, serum levels of antibodies against MMP1, CBX1, and CBX5 could potentially serve as useful tools for diagnosing TIA and predicting the onset of aCI.

A novel nucleic acid analogue shows strong angiogenic activity.

A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100muM was stronger than that of vascular endothelial growth factor (VEGF), a positive control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.

Dipalmitoylation of radicicol results in improved efficacy against tumor growth and angiogenesis in vivo.

Tumor-related angiogenesis is likely to be a potential target for the treatment of cancer. One key to develop this angiostatic strategy would be to find useful angiogenesis inhibitors. Here we report the effects of radicicol, a microbial angiogenesis inhibitor that we previously identified using the chorioallantoic membrane assay, and its novel analog, 14,16-dipalmitoyl-radicicol, on tumor angiogenesis and growth. As expected for agents containing a penolic hydroxyl group, systemic administration of radicicol had little or no effect on neovascularization triggered by a M5076 mouse tumor cell line or a RMT-1 rat mammary carcinoma cell line established from autochthonous rat mammary tumors induced by 7,12-dimethylbenz[a]anthracene in a mouse dorsal air sac assay system. The agent did not show growth-inhibitory activity against either transplantable M5076 tumors or autochthonous 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors. In contrast, 14,16-dipalmitoyl-radicicol potently suppressed tumor angiogenesis and growth in these experimental models. Furthermore, the analog significantly prolonged the survival rate of M5076-implanted mice. Although not stronger than radicicol, it dose-dependently inhibited embryonic angiogenesis in the chorioallantoic membrane assay, the dose required for half-maximal inhibition (ID(50)) value being 23 microg (27 nmol) per egg, and showed concentration-dependent antiproliferative activity against microvascular endothelial cells in vitro. These data suggest that 14,16-dipalmitoyl-radicicol is a promising antitumor agent with antiangiogenic activity.

Bovine lactoferrin inhibits tumor-induced angiogenesis.

Recent studies have demonstrated that bovine lactoferrin (bLF) suppresses tumor growth and metastasis in the mouse and rat and moreover may inhibit angiogenesis. To determine whether angiogenesis inhibition might contribute to antitumor activity, we examined the influence of bLF on tumor-induced angiogenesis and endothelial cell functions as well as angiogenesis-related cytokine production. Bovine LF exhibited dose-dependent inhibition of angiogenesis on 4-6-day-old chick embryo chorioallantoic membranes (CAMs) that lack a mature immune response. This inhibition was reversed when bLF was simultaneously treated with basic fibroblast growth factor (bFGF). It also inhibited in vitro formation of tube-like structures of mouse endothelial KOP2.16 cells. Moreover, it potently suppressed bFGF- or VEGF-induced proliferation of mouse endothelial KOP2.16 cells, but not of mouse fibroblast A31 cells and Lewis lung carcinoma (3LL) cells. In mice, both orally and intraperitoneally administered bLF significantly and dose-dependently suppressed 3LL cell-induced angiogenesis in a dorsal air sac assay. As orally administered bLF was reported to exhibit antitumor activity through production of interferon (IFN)-gamma and interleukin (IL)-18 in intestinal mucosa (Kuhara T et al., Nutr Cancer 2000;38:192-9), production of these cytokines in mouse serum and peritoneal macrophages by bLF was examined. IFN-gamma was not detected in serum by bLF administration. However, bLF markedly elevated IL-18 concentration in serum by oral administration, but not by intraperitoneal administration. It also induced IL-18 in peritoneal macrophages in vitro. These results suggest that bLF participates as a regulator of angiogenesis, possibly explained by blocking endothelial function and inducing IL-18 production. Antitumor activity of bLF may thus be partly mediated by angiogenesis inhibition.

Retinoic acid controls blood vessel formation by modulating endothelial and mural cell interaction via suppression of Tie2 signaling in vascular progenitor cells.

Inhibition by all-trans retinoic acid (atRA) of the microvasculature formation in chicken chorioallantoic membrane (CAM) accompanied remarkably reduced numbers of endothelial cells (ECs) and increased numbers of mural cells (MCs) under the chorionic epithelial layer. Ro41-5253 (retinoid antagonist) exerted the opposite effect. Although atRA did not affect the differentiation of murine embryonic stem cell-derived vascular progenitor cells (VPCs) into ECs or MCs, atRA suppressed EC-MC interaction, leading to impaired branching. In both atRA-treated VPC cultures and CAM tissues underneath the chorionic epithelial layer, the expression of angiopoietin-2 (Ang-2; competitor for Ang-1) was enhanced, whereas that of Tie2 (a receptor for Angs) was reduced. Simultaneous treatment with Ang-1 partially blocked RA induction of EC-MC malinteraction and reduction in blood vessel formation. These results suggest that retinoid(s) may reduce EC-MC interaction by down-regulating Tie2 signaling as well as decreased EC numbers, which lead to impaired vascular remodeling.

Novel function of ascorbic acid as an angiostatic factor.

Endothelial permeability is increased by vascular endothelial cell growth factor and decreased by antioxidants. Whether or not l-ascorbic acid (Asc), which decreases endothelial permeability by stimulating the endothelial barrier function, is anti-angiogenic (angiostatic) remains unknown. We examined the role of Asc on angiogenesis using two assay systems. At first, the potential role of Asc on four steps of angiogenesis was investigated in cultured bovine microvascular endothelial cells. Asc inhibited the formation of vessel-like tubular structures of endothelial cells cultured on Matrigel; however, it did not decrease the activity of plasminogen activator (PA), which creates the space into which vascular vessels extend. Furthermore, even at high concentrations, Asc did not inhibit either the proliferation or migration of endothelial cell cultures. Secondly, whether Asc inhibited in vivo angiogenesis or not was studied on chick chorioallantoic membrane (CAM) during the 4-6 days of embryogenesis when neovascularization is rapid. It also revealed that angiogenesis was dose-dependently inhibited by Asc from 0.5 micro mol/CAM with half-maximal inhibition at 2.5 micro mol/CAM. Because it was previously reported that the endothelial barrier function decreases permeability via the stimulation of collagen synthesis induced by Asc, we treated CAM with the inhibitor of collagen synthesis, l-azetidine 2-carboxylic acid (AzC). This compound partially attenuated the angiostatic function of Asc on CAM. To understand the involvement of an antioxidant activity in the angiostatic function of Asc, we further examined the effect of glutathione (GSH), which is an endogenous antioxidant, on angiogenesis in CAM and endothelial cells. GSH inhibited CAM angiogenesis, as well as the formation of vessel-like tubular structures of endothelial cell cultures on Matrigel. Both Asc and GSH inhibited hydrogen peroxide (H(2)O(2)) induced tubular morphogenesis. These findings suggest that Asc affects angiogenesis through both its antioxidant properties and the stimulation of collagen synthesis. As the angiostatic activity of Asc may be one of the many effects involved in host resistance to the growth or invasiveness of solid cancer, it may be useful as a supplementary therapy in various angiogenic diseases.