Recombinant annexin II modulates impaired fibrinolytic activity in vitro and in rat carotid artery.

Fibrinolytic activity has been reported to be decreased in atherosclerosis. Recently, annexin II was identified as a coreceptor on endothelial cells for plasminogen and tissue plasminogen activator. In this study, we examined whether recombinant annexin II (rAN II) protein can modulate fibrinolytic activity on vascular endothelium in vitro and in vivo. The effect of rAN II on human umbilical vein endothelial cells (HUVECs) was measured. Addition of a fluorescent plasmin substrate revealed that HUVECs treated with rAN II exhibited significantly more plasmin generation than those treated with BSA. Moreover, rAN II treatment of HUVECs restored plasmin generation impaired by plasminogen activator inhibitor-1 or homocysteine pretreatment. In a rat carotid artery thrombus model, the patency of thrombosed carotid arteries was significantly enhanced by rAN II injection, in contrast to BSA injection, without systemic blood coagulation dysregulation. We found that rAN II enhanced plasmin generation on vascular endothelium in vitro and reduced thrombus formation in vivo, and concluded that enhancement of endothelial fibrinolytic activity by annexin II could modulate the hypercoagulable state of atherosclerosis. Further study of rAN II in vitro and in vivo may lead to the establishment of novel therapeutic approaches to thrombogenic vascular disease.

Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.

Neuritogenesis and the nerve growth factor-induced differentiation of PC-12 cells requires annexin II-mediated plasmin generation.

One of the key morphological changes associated with the nerve growth factor (NGF)-induced differentiation of rat adrenal pheochromocytoma (PC-12) cells is the growth of axon-like processes called neurites. A growing body of evidence suggests that this process may be dependent upon plasmin, a serine protease generated from plasminogen (Plg) by either urokinase Plg activator (u-PA) or tissue Plg activator (t-PA). Prior work in our laboratory has identified annexin II (Ann-II) as a co-receptor for Plg and t-PA that promotes and localizes plasmin generation near the cell surface. In the present study, we report a 3-9-fold increase in Ann-II protein and message levels in NGF-treated PC-12 cells. Message stability and nuclear run-on assays suggest that this induction occurs at the level of gene transcription. Neurite outgrowth assays on and within a three-dimensional matrix demonstrate the inhibition of NGF-induced PC-12 cell differentiation by polyclonal and monoclonal antibodies directed against Ann-II as well as by the overexpression of antisense Ann-II mRNA. Neuritogenesis is also impaired by alpha(2)-plasmin inhibitor, antibodies directed against t-PA and u-PA, and epsilon-aminocaproic acid, a lysine analog that inhibits Plg activation and the binding of Plg to Ann-II. Plasmin generation assays reveal a 2-fold increase in plasmin production on NGF-treated PC-12 cells, which can be blocked by a polyclonal antibody directed against the tail region of Ann-II. From these data, we conclude that Ann-II is transcriptionally up-regulated by NGF and that Ann-II-mediated plasmin generation may play an important role during neurite development in the differentiating PC-12 cell.

Recombinant angiostatin prevents retinal neovascularization in a murine proliferative retinopathy model.

Retinal neovascularization is central to the pathogenesis of proliferative diabetic retinopathy, the leading cause of blindness among the middle-aged population. Angiostatin, a proteolytic fragment of plasminogen is one of the most promising inhibitors of angiogenesis currently in clinical trials. Here we show that recombinant angiostatin can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Because proliferative diabetic retinopathy is a recurrent disease, effective therapy will need to be sustained. Recombinant adeno-associated viruses permit long-term expression of transfected genes; however, they can only accommodate a small insert sequence. Thus, we engineered and tested a shortened recombinant angiostatin derivative containing a signal sequence to permit secretion. Recombinant protein was purified from the medium of transfected HEK293 cells and injected subcutaneously into treated animals. The retinal vasculature was analyzed in retinal flat mounts and using immunohistochemically stained sections. Both methods demonstrate that this short, secreted form of angiostatin is effective in reducing the development of blood vessels in a nontumor environment and has therapeutic potential for neovascular retinopathies such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion and, possibly, age-related macular degeneration.

Plasminogen-mediated matrix invasion and degradation by macrophages is dependent on surface expression of annexin II.

Genetic evidence demonstrates the importance of plasminogen activation in the migration of macrophages to sites of injury and inflammation, their removal of necrotic debris, and their clearance of fibrin. These studies identified the plasminogen binding protein annexin II on the surface of macrophages and determined its role in their ability to degrade and migrate through extracellular matrices. Calcium-dependent binding of annexin II to RAW264.7 macrophages was shown using flow cytometry and Western blot analysis of EGTA eluates. Ligand blots demonstrated that annexin II comigrates with one of several proteins in lysates and membranes derived from RAW264.7 macrophages that bind plasminogen. Preincubation of RAW264.7 macrophages with monoclonal anti-annexin II IgG inhibited (35%) their binding of 125I-Lys-plasminogen. Likewise, plasmin binding to human monocyte-derived macrophages and THP-1 monocytes was inhibited (50% and 35%, respectively) when cells were preincubated with anti-annexin II IgG. Inhibition of plasminogen binding to annexin II on RAW264.7 macrophages significantly impaired their ability to activate plasminogen and degrade [3H]-glucosamine-labeled extracellular matrices. The migration of THP-1 monocytes through a porous membrane, in response to monocyte chemotactic protein-1, was blocked when the membranes were coated with extracellular matrix. The addition of plasminogen to the monocytes restored their ability to migrate through the matrix-coated membrane. Preincubation of THP-1 monocytes with anti-annexin II IgG inhibited (60%) their plasminogen-dependent chemotaxis through the extracellular matrix. These studies identify annexin II as a plasminogen binding site on macrophages and indicate an important role for annexin II in their invasive and degradative phenotype.

A mediator of cell surface-specific plasmin generation.

It has become increasingly evident that the generation of cell surface proteases including plasmin is fundamental to a wide variety of in vivo biological processes. Cell surface receptors allow for specific controlled proteolysis, provide protection from inhibitors, and enhance catalytic efficiency. Here we describe one such receptor, annexin II, which serves as a coreceptor for tissue plasminogen activator and plasminogen and is found on a wide variety of cell types including endothelial cells, some tumor cells, monocytes and macrophages, and neuronal cells. Evidence indicates that annexin II may be crucial to the efficient generation of cell surface plasmin, endothelial cell formation of new blood vessels, and maintenance of vascular patency. Additionally, it has been shown that annexin II expression in acute promyelocytic leukemia contributes to the bleeding diathesis seen in this disease and that inhibition of annexin II may be an important mechanism in the formation of atherosclerotic plaque. Furthermore, emerging evidence reveals the importance of annexin II on the surface of monocytes and macrophages, where it may contribute to the cells' ability to degrade extracellular matrix proteins and migrate to sites of injury or inflammation.

Potential role of recombinant annexin II in diabetic vascular injury.

Hyperinsulinemia and hyperglycemia have been associated with vascular injury such as atherosclerosis in diabetes mellitus. Recently, annexin II, a member of annexin family proteins, has been found to work as co-receptor on endothelial cells for plasminogen and tissue plasminogen activator, facilitating plasmin generation on the surface of vascular endothelium. In this review, we overviewed the effect of glucose and insulin on plasmin generation in endothelial cells and its potential modulation by recombinant annexin II (rAN II) based on our data.

New concepts in fibrinolysis and angiogenesis.

Angiogenesis, the process by which new blood vessels form from preexisting vasculature, underlies a number of biologic processes including embryologic development, inflammation, wound healing, hypoxic retinal vascular proliferation, tumor growth, and atherosclerosis. The fibrinolytic system represents a cascade of serine protease activation events that culminate in the generation of plasmin. Although in-vitro studies suggest several possible roles that plasmin might play in angiogenesis, angiogenesis and fibrinolytic activity do not always correlate in in-vivo systems. During cutaneous and corneal wound healing, for example, angiogenesis proceeds normally in plasminogen-deficient animals. Similarly, the growth of most neoplasms is unimpaired in the absence of plasminogen. On the other hand, hypoxia-driven vascular proliferation may require plasmin-like activity, and angiogenesis within the atherosclerotic plaque seems to be associated with increased expression of fibrinolytic proteins. Recently, several nonplasmin fibrinolysins that may support the invasive phenotype of endothelial cells under specific circumstances have been identified. Thus, the contribution of individual fibrinolysins appears to be context-specific, just as the profile of endothelial cell gene expression depends upon the surrounding tissue milieu.

Annexin II and regulation of cell surface fibrinolysis.

The regulated function of the fibrinolytic system is fundamental to the solubilization of fibrin-containing thrombi and to a number of other biologic processes. In recent years, several receptors, which serve to localize proteolytic activity on the cell surface, have been identified on endothelial cells, blood cells, neuronal cells, and tumor cells. One such receptor is annexin II, a calcium- and phospholipid-binding protein that serves as a profibrinolytic co-receptor for tissue plasminogen activator and plasminogen on endothelial cells. Accumulating evidence suggests that impaired cell surface fibrinolytic assembly could lead to progressive atherothrombotic disease. In addition, dysregulation of annexin II expression in acute promyelocytic leukemia is an important mechanism for the bleeding diathesis associated with this malignancy.

Construction of recombinant adenoviral vector of annexin II.

Annexin II is a member of the annexin family of calcium-dependent phospholipid binding proteins expressed in vascular endothelium. Recently this molecule was reported to play a role in control of fibrinolysis on the endothelial surface. To examine the role of annexin II in vascular endothelium critically, we developed a recombinant adenoviral vector containing the annexin II cDNA. A full-length annexin II cDNA was inserted into a shuttle vector, pAdRSV4, and co-transfected into 293 cells with a replication-deficient type 5 adenovirus, pJM17. Resulting plaques were isolated and checked for protein expression. The verified clone (AdRSV-ANII) was further analyzed. Characterization of this vector will facilitate the investigation of the mechanism of fibrinolysis on vascular endothelium.

High affinity binding of beta 2-glycoprotein I to human endothelial cells is mediated by annexin II.

Beta(2)-glycoprotein I (beta(2)GPI) is an abundant plasma phospholipid-binding protein and an autoantigen in the antiphospholipid antibody syndrome. Binding of beta(2)GPI to endothelial cells targets them for activation by anti-beta(2)GPI antibodies, which circulate and are associated with thrombosis in patients with the antiphospholipid antibody syndrome. However, the binding of beta(2)GPI to endothelial cells has not been characterized and is assumed to result from association of beta(2)GPI with membrane phospholipid. Here, we characterize the binding of beta(2)GPI to endothelial cells and identify the beta(2)GPI binding site. (125)I-beta(2)GPI bound with high affinity (K(d) approximately 18 nm) to human umbilical vein endothelial cells (HUVECs). Using affinity purification, we isolated beta(2)GPI-binding proteins of approximately 78 and approximately 36 kDa from HUVECs and EAHY.926 cells. Amino acid sequences of tryptic peptides from each of these were identical to sequences within annexin II. A role for annexin II in binding of beta(2)GPI to cells was confirmed by the observations that annexin II-transfected HEK 293 cells bound approximately 10-fold more (125)I-beta(2)GPI than control cells and that anti-annexin II antibodies inhibited the binding of (125)I-beta(2)GPI to HUVECs by approximately 90%. Finally, surface plasmon resonance studies revealed high affinity binding between annexin II and beta(2)GPI. These results demonstrate that annexin II mediates the binding of beta(2)GPI to endothelial cells.

Inhibition of endothelial cell thromboresistance by homocysteine.

Homocysteine (HC) is a highly reactive thiol intermediate in amino acid metabolism, which can modify the function of endothelial cells in a myriad of ways. In vitro, homocysteine can inhibit the thromboresistance properties of the endothelial cell by induction of procoagulant factors, inactivation of natural anticoagulant systems, and suppression of vasodilatory and platelet-modulating factors. HC also inhibits the fibrinolytic system by impairing the ability of the endothelial cell to bind tissue plasminogen activator (t-PA), by interacting directly with the t-PA binding "tail" domain of its endothelial cell receptor, annexin II. Moreover, HC influences endothelial cell gene expression as exemplified by induction of the elongation factor-1 family of polypeptides, which promote polypeptide chain elongation during mRNA translation. Induction of EF-1 subunits alpha, beta, gamma and delta by homocysteine is associated with increased turnover of at least one free thiol-containing protein, suggesting that up-regulation of these subunits may represent a mechanism for replacement of damaged or modified proteins. A more complete understanding of the diverse effects of homocysteine on endothelial cell function may provide important clues to the precise role homocysteine may play in the initiation and progression of vascular disease.

Simplified production of a recombinant human angiostatin derivative that suppresses intracerebral glial tumor growth.

Angiostatin is an endogenous inhibitor of tumor neovascularization that inhibits the proliferation of endothelial cells. Production of sufficient quantities of biologically active angiostatin by the enzymatic cleavage of plasminogen has proven difficult in that it has delayed clinical testing. We have cloned, expressed, and purified a recombinant human angiostatin derivative (K1-3) using a mammalian expression system. Through the addition of a secretory signal and polyhistidine sequence tag, K1-3 can be purified from post-culture medium by simple column chromatography. Purified K1-3 protein is apparently folded in an active conformation, as evidenced by its ability to bind to lysine-Sepharose. In vitro, recombinant K1-3 significantly suppressed endothelial cell proliferation in a dose-dependent manner with an IC50 of 50 nM. Using an animal model of intracranial brain tumors in immune-competent rats, systemic administration of purified recombinant K1-3 resulted in up to 85% suppression of tumor growth (P = 0.011). Growth suppression was accompanied by a 32% decrease (P = 0.01) in tumor neovascularization. This study demonstrates a simple method to produce a biologically active recombinant angiostatin derivative. The ability to suppress intracerebral tumor growth after systemic administration suggests that K1-3 is likely to have therapeutic value in the treatment of malignant glial tumors.

Annexin II and bleeding in acute promyelocytic leukemia.

BACKGROUND: Acute promyelocytic leukemia (APL) is associated with a hemorrhagic disorder of unknown cause that responds to treatment with all-trans-retinoic acid. METHODS: We studied a newly described receptor for fibrinolytic proteins, annexin II, in cells from patients with APL or other leukemias. We examined initial rates of in vitro generation of plasmin by tissue plasminogen activator (t-PA) in the presence of APL cells that did or did not have the characteristic translocation of APL, t(15;17). We also determined the effect of all-trans-retinoic acid on the expression of annexin II and the generation of cell-surface plasmin. RESULTS: The expression of annexin II, as detected by a fluorescein-tagged antibody, was greater on leukemic cells from patients with APL than on other types of leukemic cells (mean fluorescence intensity, 6.9 and 2.9, respectively; P<0.01). The t(15;17)-positive APL cells stimulated the generation of cell-surface, t-PA-dependent plasmin twice as efficiently as the t(15;17)-negative cells. This increase in plasmin was blocked by an anti-annexin II antibody and was induced by transfection of t(15;17)-negative cells with annexin II complementary DNA. The t(15;17)-positive APL cells contained abundant messenger RNA for annexin II, which disappeared through a transcriptional mechanism after treatment with all-trans-retinoic acid. CONCLUSIONS: Abnormally high levels of expression of annexin II on APL cells increase the production of plasmin, a fibrinolytic protein. Overexpression of annexin II may be a mechanism for the hemorrhagic complications of APL.

Annexin II: a mediator of the plasmin/plasminogen activator system.

The annexins constitute a family of calcium-dependent membrane binding proteins. Recently, annexin II has been shown to accelerate the activation of the clot-dissolving protease plasmin by complexing with the plasmin precursor plasminogen and with tissue plasminogen activator. Binding of plasminogen to annexin II is inhibited by the atherogenic lipoprotein, lipoprotein(a), while binding of tissue plasminogen activator to annexin II is blocked by the thiol amino acid homocysteine. Formation of the plasminogen/tissue plasminogen activator/annexin II complex may represent a key regulatory mechanism in fibrinolytic surveillance.

Modulation of annexin II by homocysteine: implications for atherothrombosis.

Recent evidence indicates a potential role for the plasmin/plasminogen activator system in the prevention of atherosclerotic vascular disease. Fibrin deposition is a common histologic feature of the tissues of mice that are genetically deficient in one or more key components of the fibrinolytic system. Cell surface receptors may support fibrinolytic surveillance in both intravascular and extravascular locations by stimulating the efficiency plasmin generation and by protecting plasmin from its inhibitors. In vitro studies suggest that the endothelial cell receptor, annexin II, which independently binds both plasminogen and t-PA, could play a key role in the process. Binding of plasminogen to annexin II is specifically inhibited in the presence of excess concentrations of the atherogenic LDL-like particle Lp(a). Similarly, t-PA binding to annexin II is blocked by homocysteine, a sulfhydryl-containing amino acid that is associated with atherogenesis and that directly derivatizes the t-PA binding domain of annexin II. Elucidation of the precise role of annexin II in fibrinolytic surveillance, however, will await in vivo study.

Induction of acute translational response genes by homocysteine. Elongation factors-1alpha, -beta, and -delta.

The thiol amino acid homocysteine (HC) accumulates in homocystinuria and homocyst(e)inemia, and is associated with a wide variety of clinical manifestations. To determine whether HC influences the cell's program of gene expression, vascular endothelial cells were treated with HC for 6-42 h and analyzed by differential display. We found a 3-7-fold, time-dependent induction of a 220-base pair fragment, which demonstrated complete sequence identity with elongation factor-1delta (EF-1delta), a member of the multimeric complex regulating mRNA translation. Fibroblasts from cystathionine beta-synthase -/- individuals also showed up to 3.0-fold increased levels of mRNA for EF-1alpha, -beta, and -delta when compared with normal cells, and treatment of normal cells with the HC precursor, methionine, induced a 1.5-2.0-fold increase in EF-1alpha, -beta, and -delta mRNA. This induction was completely inhibited by cycloheximide and reflected a doubling in the rate of gene transcription in nuclear run-on analyses. In HC-treated endothelial cells, pulse-chase studies revealed a doubling in the rate of synthesis of the thiol-containing protein, annexin II, but no change in synthesis of the cysteineless protein, plasminogen activator inhibitor-1. Thus, HC induces expression of a family of acute translational response genes through a protein synthesis-dependent transcriptional mechanism. This process may mediate accelerated synthesis of free thiol-containing proteins in response to HC-induced oxidative stress.

Profibrinolytic properties characterize a stably transformed human endothelial cell line.

A stable immortalized venous endothelial cell (IVEC) line, obtained by transfection of human umbilical vein endothelial cells (HUVEC), retains many normal differentiated endothelial characteristics. We compared the fibrinolytic activities of IVEC and HUVEC, and observed that IVEC express a more profibrinolytic phenotype than HUVEC, since they bind and activate plasminogen more efficiently, produce more tissue plasminogen activator and urokinase-type plasminogen activator antigens, and secrete less plasminogen activator inhibitor-1 antigen both under basal conditions and after stimulation with lipopolysaccharide, phorbol ester and tumor necrosis factor. Moreover, immunostaining and Western blotting of IVEC for the plasminogen/tissue plasminogen activator receptor annexin II, as well as Northern blotting of annexin II mRNA, revealed similar patterns of surface expression in IVEC and HUVEC. Plasminogen activator inhibitor-2 is expressed similarly in both cell types. IVEC may be a useful human model for functional and pharmacological explorations and modulations of fibrinolytic system components.

Tissue plasminogen activator binding to the annexin II tail domain. Direct modulation by homocysteine.

Tissue plasminogen activator binds to endothelial cells via the calcium-regulated phospholipid-binding protein annexin II, an interaction that is inhibited by the prothrombotic amino acid homocysteine. We sought to identify the tissue plasminogen activator binding domain of annexin II and to determine the mechanism of its modulation by homocysteine. Tissue plasminogen activator binding to immobilized annexin II was inhibited by intact fluid phase annexin II but not by its "core" fragment (residues 25-339). Two overlapping "tail" peptides specifically blocked 65-75% of binding. Localization of the tissue plasminogen activator binding domain was confirmed upon specific inhibition by the hexapeptide LCKLSL (residues 7-12). Expressed C9G annexin II protein failed to support tissue plasminogen activator binding, while binding to C133G, C262G, and C335G was equivalent to that of wild type annexin II. Upon exposure to homocysteine, annexin II underwent a 135 +/- 4-Da increase in mass localizing specifically to Cys9 and a 60-66% loss in tissue plasminogen activator-binding capacity (I50 = 11 microM). Upon treatment of cultured endothelial cells with [35S]homocysteine, the dithiothreitol-sensitive label was recovered by immunoprecipitation with anti-annexin II IgG. These data provide a potential mechanism for the prothrombotic effect of homocysteine by demonstrating direct blockade of the tissue plasminogen activator binding domain of annexin II.