Procoagulant and fibrinolytic activity after polytrauma in rat.

The purpose of this study was to determine whether trauma-induced coagulopathy is due to changes in 1) thrombin activity, 2) plasmin activity, and/or 3) factors that stimulate or inhibit thrombin or plasmin. Sprague-Dawley rats were anesthetized with 1-2% isoflurane/100% oxygen, and their left femoral artery and vein were cannulated. Polytrauma included right femur fracture, and damage to the small intestines, the left and medial liver lobes, and right leg skeletal muscle. Rats were then bled 40% of blood volume. Plasma samples were taken before trauma, and at 30, 60, 120, and 240 min. Polytrauma and hemorrhage led to a significant fall in prothrombin levels. However, circulating thrombin activity did not change significantly over time. Antithrombin III and α2 macroglobulin fell significantly by 2 h, then rose by 4 h. Soluble thrombomodulin was significantly elevated over the 4 h. Circulating plasmin activity, plasminogen, and D-dimers were elevated for the entire 4 h. Tissue plasminogen activator (tPA) was elevated at 30 min, then decreased below baseline levels after 1 h. Plasminogen activator inhibitor-1 was significantly elevated at 2-4 h. Neither tissue factor pathway inhibitor nor thrombin activatable fibrinolysis inhibitor changed significantly over time. The levels of prothrombin and plasminogen were 30-100 times higher than their respective active enzymes. Polytrauma and hemorrhage in rats lead to a fibrinolytic coagulopathy, as demonstrated by an elevation in plasmin activity, D-dimers, and tPA. These results are consistent with the observed clinical benefit of tranexamic acid in trauma patients.

High-level expression, purification, and enzymatic characterization of truncated human plasminogen (Lys531-Asn791) in the methylotrophic yeast Pichia pastoris.

BACKGROUND: Plasmin is a serine protease that plays a critical role in fibrinolysis, which is a process that prevents blood clots from growing and becoming problematic. Recombinant human microplasminogen (rhµPlg) is a derivative of plasmin that solely consists of the catalytic domain of human plasmin and lacks the five kringle domains found in the native protein. Developing an industrial production method that provides high yields of this protein with high purity, quality, and potency is critical for preclinical research. RESULTS: The human microplasminogen gene was cloned into the pPIC9K vector, and the recombinant plasmid was transformed into Pichia pastoris strain GS115. The concentration of plasmin reached 510.1 mg/L of culture medium. Under fermentation conditions, the yield of rhµPlg was 1.0 g/L. We purified rhµPlg to 96% purity by gel-filtration and cation-exchange chromatography. The specific activity of rhµPlg reached 23.6 U/mg. The K m of substrate hydrolysis by recombinant human microplasmin was comparable to that of human plasmin, while rhµPlm had higher k cat /Km values than plasmin. The high purity and activity of the rhµPlg obtained here will likely prove to be a valuable tool for studies of its application in thrombotic diseases and vitreoretinopathies. CONCLUSIONS: Reliable rhµPlg production (for use in therapeutic applications) is feasible using genetically modified P. pastoris as a host strain. The successful expression of rhµPlg in P. pastoris lays a solid foundation for its downstream application.

Shark cartilage extract induces cytokines expression and release in endothelial cells and induces E-selectin, plasminogen and t-PA genes expression through an antioxidant-sensitive mechanism.

Neovastat® is a standardized extract of marine cartilage, an avascular tissue, which contains many biologically active molecules and has multiple antiangiogenic properties. In addition to VEGFR2 and MMPs inhibition, shark cartilage extract (SCE) has recently been shown to induce tissue plasminogen activator gene (PLAT) expression in bovine endothelial cells in a TNF like manner, by inducing the typical mediators NF-κB and JNK. There is now compelling evidences that the NF-κB and JNK pathways are activated by cytokines induced generation of reactive oxygen species (ROS). We used macroarray genes expression analysis on human umbilical vein endothelial cells, to investigate if that mechanism could mediate the effect of SCE. Transcriptomic results showed that SCE induced expression of several cytokines. Their impact must be important, given that treatment of endothelial cells with the cytokine TNF-α was able to reproduce most of the effects of cartilage extract on genes expression. In addition, most of the genes, known to be inducible by NF-κB or JNK following cytokines stimulation, were less induced by SCE when endothelial cells were pretreated with the antioxidant N-Acetylcysteine (NAC), suggesting a role of ROS in endothelial cell activation by SCE. Finally, the possible effects of PLAT, PLG, SELE, IL8 and PRDX2 (those validated by q-PCR) on angiogenesis, will also be discussed.

Induction of macrophage plasminogen activator by endotoxin stimulation and phagocytosis: evidence for a two-stage process.

The injection of thioglycollate medium into the peritoneal cavity of the mouse induces high levels of macrophage fibrinolytic activity due to the production and secretion of a plasminogen activator, a trypsinlike serine protease, which is absent in unstimulated macrophages. Intraperitoneal injection of endotoxin or mineral oil can stimulate only a fraction (<10%) of the fibrinolytic activity of thioglycollate cells, similar to the partial stimulation (<10%) seen 1-2 days after phagocytosis of latex or SRBC by unstimulated macrophages. The endotoxin-stimulated macrophages contain and release relatively low levels of plasminogen activator, but these primed cells can be triggered to produce and secrete high levels of enzyme, by phagocytosis of latex. Under conditions where the plasminogen activator is induced and secreted, there are no effects on the production and/or release of lysozyme or intracellular acid hydrolases, Discovery of a two-stage procedure for inducing macrophage plasminogen activator made it possible to study the role of cell priming and phagocytosis separately. Endotoxin was a more effective priming agent, weight for weight, than lipid A:BSA complex. Secretion of the plasminogen activator was induced only by thioglycollate, or endotoxin and latex. In situ fibrinolysis was induced by these agents and mineral oil, BCG, and fetal calf serum, in decreasing order of effectiveness. Phagocytosis of latex in all cases except thioglycollate stimulation, increased fibrinolytic activity from three- to sixfold. Latex and a variety of other particles such as M. lysodeikticus, aggregated gamma-globulin and immune complexes showed dose-dependent stimulation of fibrinolysis by endotoxin-primed macrophages. Although the initial phagocytic trigger was not specific for the substance employed, the ability to induce a sustained response depended on the persistence of the phagocytized particle within the cell. Fibrinolysis and secretion of plasminogen activator continued at high levels for at least 9 days after uptake of latex, a nondigestible particle, whereas plasminogen activator was secreted only transiently after ingestion of rapidly digested M. lysodeikticus. The induction of plasminogen activator secretion provides a mechanism by which the activated macrophage can exert a selective effect on its extracellular environment.

Preparation and characterization of RGD tumour-homing-peptide-modified plasminogen K5.

Plasminogen K5 (kringle 5) has strong inhibitory effects on endothelial-cell proliferation and migration. It was reported that K5 can reduce tumour neovascularization, resulting in clinically relevant antitumour effects. To determine whether addition of a tumour-targeting peptide could improve the tumour homing and antitumour activities of K5, we genetically modified K5 with an RGD (Arg-Gly-Asp) motif, which is a ligand with high affinity for αvß3 and αvß5 integrins. The fusion protein RGD-K5 was expressed in the Pichia pastoris system and the biological activity of RGD-K5 was assessed in vitro and in vivo. The results showed that the RGD-K5 exhibited a more potent effect of inhibiting endothelial cell proliferation and migration compared with that of traditional K5. RGD-K5 also displayed stronger anti-angiogenic activity in a CAM (chick chorioallantoic membrane) assay. Furthermore, RGD-K5 also showed stronger anti-angiogenic and antitumour effects in B16F10 melanoma-bearing mice compared with traditional K5. In conclusion, the biological activity of K5 can be further improved by the addition of a tumour-homing peptide, and the RGD-K5 may prove to be a promising novel candidate for cancer therapy.

Plaminogen is synthesized by primary cultures of rat hepatocytes.

The accumulation of rat plasminogen in the medium of primary monolayer cultures of adult parenchymal hepatocytes was detected with a quantitative immunological assay. These primary cultures synthetisized and secreted both circulating isozymic forms of plasminogen at rates sufficient to account for the majority of the in vivo plasminogen turnover.

Kidney: primary source of plasminogen after acute depletion in the cat.

The kidney was the primary source of plasminogen to restore normal plasma levels, after acute plasminogen depletion was produced by injection of streptokinase in cats. The concentration of plasminogen in the hepatic vein remained below that in the artery during the time when concentrations in the artery and renal vein were returning to normal.

Synthesis of human plasminogen by the liver.

Genetic types of plasminogen were determined from a donor and a recipient before and after hepatic homotransplantation. Examination of the plasminogen types demonstrated that the liver is the principal site of synthesis of human plasminogen.

Monoclonal antibody to cytokeratin VKIALEVEIATY sequence motif reduces plasminogen activation in breast tumour cells.

Cytokeratins (CKs) are the main structural proteins of epithelial cells. Although they mainly form cytoplasmic structures, they are also localized at the plasma membrane or secreted from the cells. Some CKs are over-expressed in tumour cells and are used as diagnostic and prognostic biomarkers. A stable hybridoma cell line producing anti-cytokeratin monoclonal antibody (anti-CK MAb) was prepared after immunizing mice with breast cancer MCF-7 cell lysate. As shown by 2D electrophoresis, immunoblotting and mass spectroscopy, the monoclonal antibody recognizes an epitope on CK1, CK2, CK8, CK10 and CK18 in MCF-7 cells. To identify the binding site of the antibody three peptides of 12 amino acids were synthesized, each overlapping a 27 amino acid consensus sequence of the recognized CKs. Anti-CK MAb expressed high affinity for a dodecapeptide with the sequence VKIALEVEIATY, localized in the CK alpha-helical B2 domain, as shown by ELISA and surface plasmon resonance. Treatment of MCF-7 cells by anti-CK MAb impaired plasminogen activation and consequently invasiveness of the cells. Our results show that, besides their use in diagnosis, anti-cytokeratin antibodies could be used in therapy of invasive breast cancer.

Development of gamma G, gamma A, gamma M, beta IC-beta IA, C 1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, alpha 1-antitrypsin, orosomucoid, beta-lipoprotein, alpha 2-macroglobulin, and prealbumin in the human conceptus.

The synthesis of gammaG, gammaA, gammaM, beta(1C)/beta(1A), C'1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, alpha(1)-antitrypsin, orosomucoid, beta-lipoprotein, alpha(2)-macroglobulin, and prealbumin was studied in 15 normal human embryos and fetuses of 29 days to 18 wk gestation and in the yolk sacs of four embryos from 5.5 to 11.5 wk gestation using tissue culture in (14)C-labeled amino acids followed by radioimmunoelectrophoresis. The human embryo as early as 29 day gestation synthesized beta(1C)/beta(1A), C'1 esterase inhibitor, transferrin, hemopexin, alpha(1)-antitrypsin, beta-lipoprotein, alpha(2)-macroglobulin, and prealbumin in culture. At 32 days gestation ceruloplasmin and orosomucoid were also synthesized, but synthesis of fibrinogen was not observed before 5.5 wk. Synthesis of gammaM occurred as early as 10.5 wk gestation, and gammaG synthesis was found in cultures as early as 12 wk gestation; gammaA synthesis was not detected in any of the tissue cultures. With the exception of the gamma-globulins, each of the proteins studied was synthesized by the liver, but additional sites of synthesis for some of these proteins were also found. Synthesis of gammaG and gammaM occurred primarily in the spleen, but other sites of synthesis were noted as well. Changes in the concentrations of most of these proteins and plasminogen in embryonic and fetal serum from 5.5 to 41 wk gestation, in amniotic fluid from 6.5 to 38 wk gestation, and in the sera of neonates during the 1st 3 wk postpartum are described. Although gammaA, gammaM, ceruloplasmin, or haptoglobin were not detectable in some of the embryonic and fetal sera, gammaA and ceruloplasmin were both present as early as 6.5 wk gestation, haptoglobin by 9.5 wk gestation, and gammaM by 17 wk gestation. Each of the other proteins were present in all of the sera examined.

Plasminogen kringle 5-engineered glioma cells block migration of tumor-associated macrophages and suppress tumor vascularization and progression.

Angiostatin, a well-characterized angiostatic agent, is a proteolytic cleavage product of human plasminogen encompassing the first four kringle structures. The fifth kringle domain (K5) of human plasminogen is distinct from angiostatin and has been shown, on its own, to act as a potent endothelial cell inhibitor. We propose that tumor-targeted K5 cDNA expression may act as an effective therapeutic intervention as part of a cancer gene therapy strategy. In this study, we provide evidence that eukaryotically expressed His-tagged human K5 cDNA (hK5His) is exported extracellularly and maintains predicted disulfide bridging conformation in solution. Functionally, hK5His protein produced by retrovirally engineered human U87MG glioma cells suppresses in vitro migration of both human umbilical vein endothelial cells and human macrophages. Subcutaneous implantation of Matrigel-embedded hK5His-producing glioma cells in nonobese diabetic/severe combined immunodeficient mice reveals that hK5His induces a marked reduction in blood vessel formation and significantly suppresses the recruitment of tumor-infiltrating CD45+ Mac3+ Gr1- macrophages. Therapeutically, we show in a nude mouse orthotopic brain cancer model that tumor-targeted K5 expression is capable of effectively suppressing glioma growth and promotes significant long-term survival (>120 days) of test animals. These data suggest that plasminogen K5 acts as a novel two-pronged anticancer agent, mediating its inhibitory effect via its action on host-derived endothelial cells and tumor-associated macrophages, resulting in a potent, clinically relevant antitumor effect.

Multiparity upregulates placental plasminogen and urokinase-type plasminogen activator.

PROBLEM: Multiparity increased the number of trophoblast cells in decidua of both low and high fetal loss mouse models. However, they differ in fetal survival rate and maternal thymocyte subpopulations, suggesting that trophoblast invasiveness is not equivalent. Our aim was to explore the involved mechanism. METHOD OF STUDY: We studied placentae from primiparous and multiparous females of low and high fetal loss models. We investigated invasiveness in vitro, expression of plasminogen, and its activators: tissue type (tPA)-urokinase type (uPA), and activity and expression of matrix metalloproteinases (MMP)-2 and MMP-9. RESULTS: Placental invasiveness is upregulated by multiparity, but lesser in the high fetal loss model. Multiparous animals showed elevated expression of plasminogen and uPA. However, the high fetal loss combination showed higher expression of a short and less active fragment of uPA (LMW-uPA). MMP-2, MMP-9, and tPA were unaffected. CONCLUSION: uPA would participate in the increased multiparity-associated placental invasiveness.

Novel Thrombolytic Drug Based on Thrombin Cleavable Microplasminogen Coupled to a Single-Chain Antibody Specific for Activated GPIIb/IIIa.

BACKGROUND: Thrombolytic therapy for acute thrombosis is limited by life-threatening side effects such as major bleeding and neurotoxicity. New treatment options with enhanced fibrinolytic potential are therefore required. Here, we report the development of a new thrombolytic molecule that exploits key features of thrombosis. We designed a recombinant microplasminogen modified to be activated by the prothrombotic serine-protease thrombin (HtPlg), fused to an activation-specific anti-glycoprotein IIb/IIIa single-chain antibody (SCE5), thereby hijacking the coagulation system to initiate thrombolysis. METHODS AND RESULTS: The resulting fusion protein named SCE5-HtPlg shows in vitro targeting towards the highly abundant activated form of the fibrinogen receptor glycoprotein IIb/IIIa expressed on activated human platelets. Following thrombin formation, SCE5-HtPlg is activated to contain active microplasmin. We evaluate the effectiveness of our targeted thrombolytic construct in two models of thromboembolic disease. Administration of SCE5-HtPlg (4 µg/g body weight) resulted in effective thrombolysis 20 minutes after injection in a ferric chloride-induced model of mesenteric thrombosis (48±3% versus 92±5% for saline control, P<0.01) and also reduced emboli formation in a model of pulmonary embolism (P<0.01 versus saline). Furthermore, at these effective therapeutic doses, the SCE5-HtPlg did not prolong bleeding time compared with saline (P=0.99). CONCLUSIONS: Our novel fusion molecule is a potent and effective treatment for thrombosis that enables in vivo thrombolysis without bleeding time prolongation. The activation of this construct by thrombin generated within the clot itself rather than by a plasminogen activator, which needs to be delivered systemically, provides a novel targeted approach to improve thrombolysis.

Analysis of the invasion-metastasis mechanism in pancreatic cancer: involvement of plasmin(ogen) cascade proteins in the invasion of pancreatic cancer cells.

To clarify the potential involvement of plasmin(ogen) cascade proteins in the cell dissociation and subsequent invasion of pancreatic cancer cells, western blot analysis, immunocytochemistry, immunohistochemistry, and in vitro invasion assay were performed in the cell lines or tissue of pancreatic cancer. The strong expression of plasmin(ogen), urokinase type plasminogen activator (uPA) and uPA receptor (uPAR), and apparently weak expression of the relevant proteins were found in the conditioned medium of dissociated (PC-1.0) and non-dissociated (PC-1) pancreatic cancer cells, respectively. Furthermore, uPA-treatment significantly induced the expression of plasmin(ogen) and uPAR in the conditioned medium of non-dissociated (PC-1) pancreatic cancer cells. Moreover, the expression of plasmin(ogen) and uPAR was stronger at the invasive front than at the center of human pancreatic cancer tissue. On the other hand, plasmin-treatment induced the expression of matrix metalloproteinase-2 (MMP-2), MMP-7 and MMP-9 in PC-1 cells. Simultaneously, plasmin- or uPA-treatments obviously induced the dissociation of cell colonies and in vitro invasiveness in PC-1 cells. The plasmin(ogen) cascade is closely involved in the invasion of pancreatic cancer cells and, especially in its early stage, cell dissociation. Targeting the plasmin(ogen) cascade may provide a new insight into molecular target therapy based on anti-invasion and anti-metastasis for pancreatic cancer.

Decorin promotes plasminogen/plasmin expression within acute spinal cord injuries and by adult microglia in vitro.

Spinal cord scar tissue presents a combined physical and molecular barrier to axon regeneration. Theoretically, spinal cord injuries (SCIs) can be rendered more permissive to axon growth by either suppressing synthesis of misaligned, fibrotic scar tissue and associated axon growth inhibitors, or enzymatically degrading them. We have previously shown that acute infusion of human recombinant decorin core protein into discreet stab injuries of the rat dorsal column pathways effected a major suppression of inflammation, astrogliosis, and multiple axon growth inhibitory chondroitin sulfate proteoglycans, which combined to promote rapid axon growth across the injury site. The high efficiency of chondroitin sulfate proteoglycan (CSPG) core protein suppression (approximately 90%) suggested that decorin may promote CSPG degradation in addition to suppressing CSPG synthesis. As the serine protease plasmin can degrade axon growth inhibitory CSPGs (neurocan and phosphacan) and its zymogen, plasmininogen is synthesized by microglia, we have investigated whether decorin treatment of acute SCIs and cultured adult spinal cord microglia can increase plasminogen/ plasmin synthesis. Infusion of hr-decorin over the first 8 days post-SCI induced 10- and 17-fold increases in plasminogen and plasmin protein levels, respectively, within sites of injury and a threefold increase in microglial plasminogen mRNA in vitro. In addition to potentially degrading multiple axon growth inhibitory components of the glial scar, plasmin is known to play major roles in activating neurotrophins and promoting central nervous system (CNS) plasticity. The wider implications of decorin induction of plasmin in the injured spinal cord for axon regeneration, and recovery of function at acute and chronic time points post-SCI are reviewed.

Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, angiostatin.

Angiostatin is an inhibitor of angiogenesis and metastatic growth that is found in tumor-bearing animals and can be generated in vitro by the proteolytic cleavage of plasminogen. The mechanism by which angiostatin is produced in vivo has not been defined. We now demonstrate that human prostate carcinoma cell lines (PC-3, DU-145, and LN-CaP) express enzymatic activity that can generate bioactive angiostatin from purified human plasminogen or plasmin. Affinity purified PC-3-derived angiostatin inhibited human endothelial cell proliferation, basic fibroblast growth factor-induced migration, endothelial cell tube formation, and basic fibroblast growth factor-induced corneal angiogenesis. Studies with proteinase inhibitors demonstrated that a serine proteinase is necessary for angiostatin generation. These data indicate that bioactive angiostatin can be generated directly by human prostate cancer cells and that serine proteinase activity is necessary for angiostatin generation.

Radiation therapy to a primary tumor accelerates metastatic growth in mice.

The surgical removal of a primary tumor can result in the rapid growth of metastases. The production of angiogenesis inhibitors by the primary tumor is one mechanism for the inhibition of metastatic tumor growth. The effect of curative radiotherapy to a primary tumor known to make an inhibitor of angiogenesis and the effects on distant metastases has not been studied. We here show that the eradication of a primary Lewis lung carcinoma (LLC-LM), which is known to generate angiostatin, is followed by the rapid growth of metastases that kill the animal within 18 days after the completion of radiation therapy. The right thighs of C57BL/6 mice (n = 25) were injected s.c. with 1 x 10(6) LLC-LM cells. Animals were randomized to one of five groups: no irradiation, 40 Gy in one fraction, 30 Gy in one fraction, 40 Gy in two 20 Gy fractions, or 50 Gy in five 10 Gy fractions. Tumors were clinically eradicated in each treatment group. All of the surviving animals became dyspneic and were killed within 14-18 days after the completion of radiation therapy. Examination of their lungs revealed >46 (range, 46-62) surface metastases in the treated animals compared with 5 (range, 2-8) in the untreated animals. The lung weights had increased from 0.2 g (range, 0.19-0.22 g) in the controls to 0.58 g (range 0.44-0.84) in the experimental animals. The most effective dose regimen was 10 Gy per fraction for five fractions, and serial experiments were conducted with this fractionation scheme. Complete response of the primary tumor was seen in 25 of 35 (71%) mice. The average weight of the lungs in the nonirradiated animals was 0.22 g (range, 0.19-0.24 g) and in the irradiated animals was 0.66 g (range, 0.61-0.70 g). The average number of surface metastases increased from five per lung (range, 2-13) in the control animals to 53 per lung (range, 46-62) in the irradiated animals. Both differences were statistically significant with P < 0.001. If the nontumor-bearing leg was irradiated or the animals were sham-irradiated, no difference in the number of surface metastases or lung weights was observed between the control group and the treated group. Urinary levels of matrix metalloproteinase 2, the enzyme responsible for angiostatin processing in this tumor model, were measured and correlated with the viability and size of the primary tumor. Administration of recombinant angiostatin prevented the growth of the metastases after the treatment of the primary tumor. In this model, the use of radiation to eradicate a primary LLC-LM tumor results in the growth of previously dormant lung metastases and suggests that combining angiogenesis inhibitors with radiation therapy may control distant metastases.

The plasminogen-plasminogen activator (PA) system in neuroblastoma: role of PA inhibitor-1 in metastasis.

Proteases of the plasminogen-plasminogen activator (PA) system play an important role in cancer metastasis. We have examined the expression of these proteases and their cell surface receptors and inhibitors in neuroblastoma, a tumor that originates in cells of the neural crest and is the second most common solid tumor in children. This analysis was performed in seven established human cell lines and 20 primary tumor specimens. Urokinase PA and, in particular, tissue-type PA were expressed in cell lines and in tumor tissues; however, their levels of expression did not correlate with clinical stage. There was little evidence suggesting that neuroblastoma cells concentrate PA activity at their cell surface because urokinase-type PA receptor mRNA was detected in two cell lines and in 5 of 20 tumor samples by reverse transcription-PCR only. PA inhibitor (PAI)-2 was absent in all cell lines and tumor tissue samples examined. However, PAI-1, which was not expressed by the cell lines, was expressed by stromal cells and, specifically, endothelial cells in tumor tissue. By extending the analysis of PAI-1 expression in 64 primary tumor specimens, we found that high PAI-1 expression paradoxically correlated with metastatic stage and tumor recurrence. In vitro experiments indicated that the expression of PAI-1 by human microvascular endothelial cells was stimulated in the presence of SK-N-BE(2) human neuroblastoma cells and neuroblastoma culture medium. Recombinant PAI-1 also promoted SK-N-BE(2) cell detachment from vitronectin and migration from vitronectin toward fibronectin. From these data, we conclude that the up-regulation of PAI-1 expression in endothelial cells may promote rather than inhibit metastasis in neuroblastoma.

Human angiostatin inhibits murine hemangioendothelioma tumor growth in vivo.

Angiostatin inhibits angiogenesis and metastatic tumor growth; however, its usefulness in treating primary nonmetastasizing tumors is less well understood. We now report the effectiveness of human angiostatin administration in a mouse hemangioendothelioma model. Human angiostatin was administered to mice with s.c. hemangioendothelioma and associated disseminated intravascular coagulopathy (Kasabach-Merritt syndrome). Angiostatin significantly reduced tumor volume in comparison to nontreated controls, increased survival, and prevented the profound thrombocytopenia and anemia of Kasabach-Merritt syndrome. Apoptosis of tumor cells was induced by angiostatin, but tumor cell proliferation was not inhibited. These data suggest angiostatin as a novel treatment for nonmetastasizing vascular tumors and for Kasabach-Merritt syndrome.

Suppression of intracranial human glioma growth after intramuscular administration of an adeno-associated viral vector expressing angiostatin.

Despite various therapeutic interventions, glioblastoma multiforme (GBM) is one of the most highly vascularized neoplasms in humans with poor prognosis. In this study, we show that a single i.m. injection of an adeno-associated viral (AAV) vector expressing angiostatin, a potent angiogenic inhibitor, effectively suppresses human glioma growth in the brain of nude mice. Approximately 40% of the tumor-bearing mice treated with AAV-angiostatin vector survived for >10 months (the duration of the experiments). In contrast, 100% of the tumor-bearing mice in the control groups, with or without i.m. injection of a control vector AAV-GFP, died because of excessive tumor burden by 6 weeks. High levels of angiostatin produced by the AAV vector were detected in blood circulation for >250 days after the one-time vector injection. The secreted angiostatin specifically targeted neovessels in the brain tumors, as evidenced by the diminished vessel densities and increased apoptosis of tumor cells surrounding these neovessels. Our study thus demonstrates that AAV-mediated antiangiogenesis gene therapy offers efficient and sustained systemic delivery of the therapeutic product, which in turn effectively suppresses glioma growth in the brain.