The accumulation of angiostatin-like fragments in human prostate carcinoma.

PURPOSE: Angiostatin, a potent inhibitor of angiogenesis and, hence, the growth of tumor cell metastasis, is generated by a proteolytic enzyme from plasminogen. However, its localization and specific enzymes have yet to be ascertained in human tissue. EXPERIMENTAL DESIGN: To elucidate the generation and the localization of angiostatin in prostate carcinoma, we examined angiostatin generation in a panel of human prostate cancer cell lines and performed immunohistochemistry with the antibodies to angiostatin and prostate-specific antigen (PSA), a potent proteolytic enzyme of angiostatin in 55 cases of prostate carcinoma. RESULTS: We demonstrated that the lysates of human prostate carcinoma cell lines could generate angiostatin-like fragments from purified human plasminogen but could not generate angiostatin in the absence of exogenous plasminogen. The fragmented proteins were reacted with the monoclonal antibody specific for plasminogen lysine-binding site 1 (LBS-1). Immunohistochemically, the intracytoplasmic immunostaining of LBS-1 was positive in 87.3% (48 of 55) of prostate carcinoma cases, and the immunostaining of miniplasminogen was negative in all cases. There was a significant relationship between the positive immunostaining of LBS-1 and Gleason score (P = 0.0007). The intracytoplasmic immunostaining of PSA was positive in 37.0% (20 of 54) of prostate carcinoma cases, but there was no significant relationship between the expression of PSA and Gleason score, or between the positive immunostaining of LBS-1 and PSA. CONCLUSIONS: These findings suggest that angiostatin is generated by prostate carcinoma cells and is accumulated within the cytoplasm. In addition, the generation of angiostatin-like fragments was correlated with tumor grade; however, PSA may not be the only enzyme for angiostatin generation in human prostate carcinoma.

Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells.

Angiostatin, a potent endogenous inhibitor of angiogenesis, is generated by cancer-mediated proteolysis of plasminogen. The culture medium of human prostate carcinoma cells, when incubated with plasminogen at a variety of pH values, generated angiostatic peptides and miniplasminogen. The enzyme(s) responsible for this reaction was purified and identified as procathepsin D. The purified procathepsin D, as well as cathepsin D, generated two angiostatic peptides having the same NH(2)-terminal amino acid sequences and comprising kringles 1-4 of plasminogen in the pH range of 3.0-6.8, most strongly at pH 4.0 in vitro. This reaction required the concomitant conversion of procathepsin D to catalytically active pseudocathepsin D. The conversion of pseudocathepsin D to the mature cathepsin D was not observed by the prolonged incubation. The affinity-purified angiostatic peptides inhibited angiogenesis both in vitro and in vivo. Importantly, procathepsin D secreted by human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that by human prostate carcinoma cells. Since deglycosylated procathepsin D from both prostate and breast carcinoma cells exhibited a similar low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in carbohydrate structures of procathepsin D molecules between the two cell types. The seminal vesicle fluid from patients with prostate carcinoma contained the mature cathepsin D and procathepsin D, but not pseudocathepsin D, suggesting that pseudocathepsin D is not a normal intermediate of procathepsin D processing in vivo. The present study provides evidence for the first time that cathepsin D secreted by human prostate carcinoma cells is responsible for angiostatin generation, thereby causing the prevention of tumor growth and angiogenesis-dependent growth of metastases.

The activity of soluble VCAM-1 in angiogenesis stimulated by IL-4 and IL-13.

IL-13 is a multifunctional lymphokine sharing a number of biological properties with IL-4. We previously observed that IL-4 shows angiogenic activities in vitro as well as in vivo. In this study we examined the effect of IL-13 on angiogenesis in vitro and in vivo and also the underlying mechanisms. Human IL-13 significantly stimulated the formation of tube-like structures in collagen gels by human microvascular endothelial cells and bovine aortic endothelial cells by about 3-fold over the controls in the absence of the cytokines. Administration of murine IL-13 led to neovascularization when implanted in the rat cornea. Coadministration of neutralizing mAb to the IL-4R inhibited both tubular morphogenesis in vitro and activation of STAT6 induced by IL-4 or IL-13. Both IL-4 and IL-13 markedly increased mRNA levels of VCAM-1 in vascular endothelial cells, and the production of the soluble form of VCAM-1 was also stimulated in response to IL-4 or IL-13. Administration of anti-VCAM-1 Ab in vitro blocked tubular morphogenesis induced by IL-4 and IL-13. Angiogenesis induced in vivo in rat cornea by IL-4 and IL-13 was also inhibited by Ab against the rat alpha4 integrin subunit. These findings suggest that angiogenesis dependent on IL-4 and IL-13 is mainly mediated through a soluble VCAM-1/alpha4 integrin pathway.

New functional aspects of cathepsin D and cathepsin E.

Cathepsin D (CD) and cathepsin E are representative lysosomal and nonlysosomal aspartic proteinases, respectively, and play an important role in the degradation of proteins, the generation of bioactive proteins, antigen processing, etc. Recenty, several lines of evidence have suggested the involvement of these two enzymes in the execution of neuronal death pathways induced by aging, transient forebrain ischemia, and excessive stimulation of glutamate receptors with excitotoxins. CD has also been shown to mediate apoptosis induced by various stimuli and p53-dependent tumor suppression. To gain more insight into in vivo functions of CD, mice deficient in this enzyme were generated. The mutant animals showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound accumulation of ceroid lipofuscin, and developed a phenotype resembling neuronal ceroid lipofucinosis, suggesting that CD is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. It has also been shown that CD molecules secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogenous inhibitor of angiogenesis, suggesting its contribution to the prevention of tumor growth and angiogenesis-dependent growth of metastases. Interestingly, pro-CD from human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that from prostate carcinoma cells. Since deglycosylated CD molecules from both carcinoma cells showed a low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in the carbohydrate structures of CD molecules between the two cell types and to contribute to their potency to prevent tumor growth and metastases.

Lipoprotein(a) induces cell growth in rat peritoneal macrophages through inhibition of transforming growth factor-beta activation.

To elucidate the atherogenicity of lipoprotein(a) (Lp(a)), we examined its growth-stimulating activity in rat resident peritoneal macrophages. When macrophages were incubated with Lp(a), cell numbers were increased 1.5-fold as compared with control macrophages. Furthermore, apolipoprotein(a) (apo(a)), a plasminogen-like glycoprotein which is covalently attached to a low density lipoprotein-like particle (Lp(a)), also induced macrophage growth, while the growth-stimulating effect of Lp(a-) was negligible. These results suggest that apo(a) plays an active role in the mitogenic activity of Lp(a). Lp(a)-induced macrophage growth was inhibited by exogenously added active transforming growth factor-beta (TGF-beta) dose-dependently, and also by the addition of plasmin, which converts latent TGF-beta to an active form. Moreover, the amounts of endogenous active TGF-beta in the medium were significantly reduced by the incubation with Lp(a). It is evident from these results that Lp(a) induces macrophage growth by inhibiting TGF-beta activation. The capacity of Lp(a) to stimulate macrophage growth shown here could be novel atherogenic function of Lp(a).

Reconstituted high density lipoprotein reduces the capacity of oxidatively modified low density lipoprotein to accumulate cholesteryl esters in mouse peritoneal macrophages.

Oxidized low density lipoprotein (ox-LDL) was incubated with discoidal complexes of apolipoprotein A-I (apo A-I) and dimyristoylphosphatidylcholine (DMPC) (DMPC/apo A-I) in a cell-free system and re-isolated on Sephacryl S-400 gel filtration chromatography. Analyses of re-isolated ox-LDL showed that apo A-I was transferred from DMPC/apo A-I to ox-LDL, which accounted for 10% of the total protein of ox-LDL. Re-isolated ox-LDL also showed a 2.2-fold increase in phospholipid and a 14% decrease in cholesterol content on an apo B basis. The electrophoretic mobility of re-isolated ox-LDL was markedly reduced almost to that of native LDL. Moreover, the amounts of re-isolated ox-LDL to be degraded by mouse peritoneal macrophages as well as the capacity of re-isolated ox-LDL to accumulate cholesteryl esters (CE) in these cells were markedly reduced (60% and 80% reduction, respectively), suggesting that the ligand activity of ox-LDL for the scavenger receptor was significantly reduced upon treatment with DMPC/apo A-I. Parallel incubation of ox-LDL with free apo A-I led to a similar incorporation of apo A-I into ox-LDL. However, it had no effects on the ligand activity of ox-LDL. Thus, it is likely that the reduction in the ligand activity of ox-LDL by DMPC/apo A-I is explained by the change in the lipid moiety (mainly phospholipid) of ox-LDL. Since discoidal high density lipoprotein (HDL) is known to occur in vivo, this phenomenon might explain one of the anti-atherogenic functions of HDL.

Intravenous injection of rabbit apolipoprotein A-I inhibits the progression of atherosclerosis in cholesterol-fed rabbits.

The effects of intravenous injection of purified rabbit apoA-I on the progression of aortic atherosclerosis in cholesterol-fed rabbits were examined. In experiment 1, 28 rabbits were equally divided into groups A and B and fed a 0.5% cholesterol diet for 90 days. For the last 30 days, group B received 40 mg apoA-I every week. The fatty streak lesions in group B (23.9 +/- 15.6%) were significantly suppressed compared with those in group A (46.0 +/- 24.9%) (P < .05). In experiment 2, 33 rabbits were divided into four groups (8 or 9 rabbits per group) and fed a 0.5% cholesterol diet. Group A was killed on day 105, while groups B, C, and D were maintained for an additional 60 days on a normal diet, during which time groups C and D received 1 mg apoA-I every other day or 40 mg apoA-I every week, respectively. The lesions in group C (70.2 +/- 15.4%) and group D (65.7 +/- 20.0%) were significantly suppressed compared with those in group B (86.2 +/- 13.7%) (P < .05) but were not reduced to the level of group A (50.0 +/- 22.9%). Although apparent regression was not observed under these conditions, the present study provided the first evidence for the antiatherogenic effect of homologous and apoA-I on the progression of atherosclerosis in cholesterol-fed rabbits.

Measurement of Lp(a) with a two-step monoclonal competitive sandwich ELISA method.

OBJECTIVE: To evaluate the results of Lipoprotein (a)[Lp(a)] measurements by a competitive two-step monoclonal enzyme-linked immuno sorbent assay method comparing them with those by a conventional ELISA. METHODS: Serum having various isoforms of Lp(a) and purified Lp(a) were assayed using the method described here and commercially available kits. The reference range was determined with the use of 324 normal subjects by means of calculation from Lp(a) results of logarithmic transformation. RESULTS: Our method takes advantage of a competitive reaction between fixed antibody and free antibody to Lp(a), having the detection range up to 1000 mg/L with the lowest detection limit of 2 mg/L. The anti-Lp(a) monoclonal antibody employed in the assay system reacts uniformly with all phenotypes of Lp(a) but showing very low cross-reactivity for plasminogen and LDL. Within-run and between-run precisions were excellent, giving CVs of 2.9 and 4.0% with mean values of 145 and 635 mg/L, respectively. In comparison of the results by our method with those by a polyclonal method (Biopool) or a monoclonal antibody method (Terumo), they correlated well; Y (our method) = 0.99 x (polyclonal method, Biopool) - 1.9, r = 0.994 (n = 60), and Y = 0.94 X(monoclonal method, Terumo) -9.8, r = 0.97 (n = 60), respectively. The reference range was 105.9 +/- 25.4 mg/L, the difference between the sexes was not significant. CONCLUSION: Our method has proven highly accurate and specific. It is applicable with auto analyzer because it does not require such a pre-dilution step as is necessary for Lp(a) determination by conventional ELISA assay. Accordingly, we can conclude that our test method is workable for both clinical laboratories and mass screening.

Comparison of monoclonal and polyclonal enzyme-linked immunoabsorbent (ELISA) assays for serum Lp(a) and differences in reactivities to Lp(a) phenotypes.

We have prepared a monoclonal antibody to Lipoprotein(a)[Lp(a)] and have used it to develop an ELISA test for assaying Lp(a) in serum. The monoclonal antibody employed in the assay system reacts uniformly with S1, S2, S3 and B phenotypes of isoforms, and no cross-reaction with plasminogen at a concentration of 100 mg/dL was observed. Results of the monoclonal ELISA assay were similar to those obtained with a polyclonal antibody ELISA method and demonstrated a correlation coefficient, r = 0.99 with the equation for the regression line: Y(proposed) = 1.06 x (polyclonal antibody reference ELISA test) = 0.36 (N = 51). Inter- and intra-assay precision(CVs) of the monoclonal ELISA assay were between 2.2-3.6% at a mean Lp(a) concentration range of 19.1-68.2 mg/dL,(N = 12). Assay results of various standards were compared by both monoclonal and polyclonal antibody ELISA tests. We observed some discrepancies between expected concentrations and the polyclonal antibody ELISA assay results, which is thought to be more uniformly reactive to the various Lp(a) phenotypes. The monoclonal antibody employed in our proposed method reacts uniformly with Lp(a) phenotypes, and the assay exhibits excellent sensitivity, specificity, and accuracy and is well suited for clinical use.

Acetylated low density lipoprotein reduces its ligand activity for the scavenger receptor after interaction with reconstituted high density lipoprotein.

Complexes of apolipoprotein A-I (apoA-I) with phospholipids are known to induce cholesterol efflux from cells. In a cholesteryl ester accumulation system in which rat peritoneal macrophages were incubated with acetylated low density lipoprotein (acetyl-LDL) and either dimyristoylphosphatidylcholine complexes (DMPC/apoA-I) or native high density lipoprotein (HDL), DMPC/apoA-I exhibited a much stronger effect than native HDL in preventing cholesteryl ester accumulation. The mechanism for this phenomenon was investigated in the present study. After 18 h incubation with DMPC/apoA-I in a cell-free system, acetyl-LDL was re-isolated from DMPC/apoA-I by Sephacryl S-300 gel filtration chromatography. Re-isolated acetyl-LDL exhibited an increase in its phospholipid content by 86% as well as a reduction in the electrophoretic mobility. Its endocytic degradation by macrophages was reduced by 60% when compared with control acetyl-LDL, suggesting a significant reduction in the ligand activity for the macrophage scavenger receptor. Transfer of apolipoproteins between acetyl-LDL and DMPC/apoA-I did not occur. These results indicate that transfer of DMPC from DMPC/apoA-I to acetyl-LDL weakens the ligand activity for the scavenger receptor due probably to a decrease in net negative charge. This study demonstrated for the first time that lipid modification (change in the lipid moiety) of acetyl-LDL can induce alteration in its apolipoprotein moiety, leading to a significant loss of its biological activity. Because discoidal HDLs are known to occur in vivo, this phenomenon may explain one of the anti-atherogenic functions of HDL in vivo.