[New aspects of stroke medicine]. / Neues aus der Schlaganfallmedizin.

Systemic thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) remains the only effective and approved medical treatment of acute ischemic stroke. Several studies have demonstrated the importance of rapid recanalization. The efficacy of thrombectomy has so far not been sufficiently shown in randomized clinical trials; therefore, inclusion of suitable patients in one of the currently ongoing randomized trials is of great importance. The early treatment with magnesium after acute ischemic stroke during the pre-hospital phase did not prove to be neuroprotective. Intermittent pneumatic compression of the lower extremities in immobilized stroke patients effectively prevents deep venous thrombosis and pulmonary embolism. In patients with lacunar stroke the combination of aspirin and clopidogrel is not superior to aspirin alone and causes more bleeding complications. The novel oral anticoagulants are superior to warfarin in secondary prevention and carry a lower risk of intracranial and systemic bleeding complications. New studies will investigate whether dabigatran or rivaroxaban are superior to aspirin in secondary prevention after cryptogenic stroke.

[Diagnostic and treatment of acute ischemic stroke]. / Akutdiagnostik und Akuttherapie des ischämischen Insultes.

The incidence of first stroke in Germany is about 200.000, most of which are ischemic. The benefit of stroke unit treatment and systemic thrombolysis has been shown in large randomized trials. Diagnostic work-up besides neurologic examination includes cerebral imaging by CT or MR imaging including angiography, ultrasound of brain supplying arteries, ECG and Holter ECG and - if indicated - transesophageal echocardiography. Aspirin is the cornerstone of early secondary prevention in the acute phase, thereafter secondary prevention is determined by stroke etiology. Carotid endarterectomy or stent-assisted angioplasty are indicated in patients with hemodynamic or arterio-arterial stroke etiologies due to high-grade carotid stenosis. For cardioembolism due to atrial fibrillation, oral anticoagulation with vitamin K-antagonists or new oral anticoagulants should be started after the acute phase. In patients with non-cardioembolic stroke etiologies, platelet inhibitors are used for secondary prevention.

[New anticoagulants for stroke prevention in atrial fibrillation]. / Neue Antikoagulanzien zur Schlaganfallprävention bei Vorhofflimmern.

Oral anticoagulation with vitamin K antagonists (warfarin, phenprocoumon) is successful in both primary and secondary stroke prevention for patients with atrial fibrillation (AF), yielding a 60-70% relative reduction in stroke risk compared with placebo and a mortality reduction of 26%. However, these agents have a number of well documented shortcomings. This review describes the current landscape and developments in stroke prevention in patients with AF with special reference to secondary prevention. A number of new drugs for oral anticoagulation that do not exhibit the limitations of vitamin K antagonists are under investigation. These include direct factor Xa inhibitors and direct thrombin inhibitors. Recent studies (RE-LY, ROCKET-AF, AVERROES, ARISTOTLE) provide promising results for these new agents including higher efficacy and significantly lower incidences of intracranial bleeding compared with warfarin. The new substances show similar results in secondary as well as in primary stroke prevention in patients with AF. The new anticoagulants add to the therapeutic options for patients with AF and offer a number of advantages over warfarin for both clinician and patient, including a favorable bleeding profile and convenience of use. Consideration of these new anticoagulants will improve clinical decision-making.

Angiogenesis and antiangiogenic therapy in non-Hodgkin's lymphoma.

Angiogenesis, the growth of new blood vessels, requires dynamic expansion, assembly and stabilization of vascular endothelial cells in response to proangiogenic stimuli. Antiangiogenic strategies have become an important therapeutic modality for solid tumors. While many aspects of postnatal pathological angiogenesis have been extensively studied in the context of nonhematopoietic neoplasms, the precise role of these processes in lymphoma pathogenesis is under active investigation. Lymphoma growth and progression is potentiated by at least two distinct angiogenic mechanisms: autocrine stimulation of tumor cells via expression of vascular endothelial growth factor (VEGF) and VEGF receptors by lymphoma cells, as well as paracrine influences of proangiogenic tumor microenvironment on both local neovascular transformation and recruitment of circulating bone marrow-derived progenitors. Lymphoma-associated infiltrating host cells including hematopoietic monocytes, T cells and mesenchymal pericytes have increasingly been associated with the pathogenesis and prognosis of lymphoma, in part providing perivascular guidance and support to neoangiogenesis. Collectively, these distinct angiogenic mechanisms appear to be important therapeutic targets in selected non-Hodgkin's lymphoma (NHL) subtypes. Understanding these pathways has led to the introduction of antiangiogenic treatment strategies into the clinic where they are currently under assessment in several ongoing studies of NHL patients.

Trace eyeblink conditioning in human subjects with cerebellar lesions.

Trace eyeblink conditioning was investigated in 31 patients with focal cerebellar lesions and 19 age-matched controls. Twelve patients presented with lesions including the territory of the superior cerebellar artery (SCA). In 19 patients lesions were restricted to the territory of the posterior inferior cerebellar artery (PICA). A 3D magnetic resonance imaging was used to determine the extent of the cortical lesion and possible involvement of cerebellar nuclei. Eyeblink conditioning was performed using a 40 ms tone as conditioned stimulus (CS) followed by a stimulus free trace-interval of 400 ms and a 100 ms air-puff as unconditioned stimulus (US). In SCA patients with lesions including parts of the cerebellar interposed nucleus trace eyeblink conditioning was significantly impaired. Pure cortical lesions of the superior cerebellum were not sufficient to reduce acquisition of trace conditioned eyeblink responses. PICA patients were not impaired in trace eyeblink conditioning. Consistent with animal studies the findings of the present human lesion study suggest that, in addition to forebrain areas, the interposed nucleus is of importance in trace eyeblink conditioning. Although cortical cerebellar areas appear less important in trace compared with delay eyeblink conditioning, the present data strengthen the view that cerebellar structures contribute to different forms of eyeblink conditioning paradigms.

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.