[Fibrinolytics: from the thrombolysis to the processes of blood vessels growth and remodeling, neurogenesis, carcinogenesis and fibrosis].

One of the most outstanding scientific achievements in the thrombolysis is the development and administration of fibrinolysin - the first Soviet drug that lyses blood clots. Intracoronary administration of fibrinolysin reduced the mortality of patients with myocardial infarction by almost 20%. For his work in this field Yevgeny Chazov was awarded the Lenin Prize in 1982. Over the next decades, under his leadership, the Cardiology Center established scientific and clinical laboratories that created new generations of drugs based on fibrinolytics for treating patients with myocardial infarction, restoration of blood flow in ischemic tissue, and also studying the mechanisms of remodeling of blood vessels involving the fibrinolysis system. It have been found new mechanisms of regulation of the navigation of blood vessels and nerves growth, tumor growth and its metastasis with the participation of the fibrinolysis system proteins. The review reports the role of the fibrinolysis system in the thrombolysis, blood vessels growth and remodeling, neurogenesis, carcinogenesis and fibrosis. The article is dedicated to the 90th anniversary of academician E.I. Chazov.

[THE METHODICAL APPROACHES TO DIAGNOSTIC OF NIGHT PAROXYSMAL HEMOGLOBINURIA].

The article presents diagnostic of night paroxysmal hemoglobinuria. The night paroxysmal hemoglobinuria is an orphan disease characterized by absence of GPI-anchor on blood cells as a result of mutation of PIG-A gene on the short arm of X-chromosome. The particular proteins bounded with GPI-anchor implement function of defense from activation of components of complement and development of membrane-attacking complex. The erythrocytes exposed to destruction in bloodstream are among the most impacted. Therefore, one of the main signs of night paroxysmal hemoglobinuria is complement-depending intravascular hemolysis which indicators for a long time played a key role in diagnostic of night paroxysmal hemoglobinuria. The actual technique of diagnostic of night paroxysmal hemoglobinuria is flow cytometry. The analysis of night paroxysmal hemoglobinuria clone is recommended to patients with hemolysis of unclear genesis, thrombosis of cerebral and abdominal veins, thrombocytopenia and macrocytosis and also patients with AA, myelodysplastic syndrome, myelofibrosis. The international protocol recommended by the International Society of Clinical Cytometry (2010) is implemented to diagnose night paroxysmal hemoglobinuria. The original technique of evaluation of reticulocytes was developed with purpose to detect night paroxysmal hemoglobinuria clone. The high correlation was substantiated between size of night paroxysmal hemoglobinuria clone measured among reticulocytes according to proposed mode and night paroxysmal hemoglobinuria clone measured among granulocytes and monocytes detected according international standardized approach.

Molecular modeling as a new approach to the development of urokinase inhibitors.

Proteolytic activity of urokinase plays an important role in negative remodeling of blood vessels, restenosis, tumor angiogenesis, and metastasizing, which necessitates the development of selective urokinase inhibitors. Using methods of computer modeling (docking, post processing, and direct docking) and quantum chemistry, we selected substances from the large compound database, analyzed their structures, and experimentally verified their inhibitor activity. New urokinase inhibitor candidates were proposed based on the theoretical predictions and experimental verification of compound activities. The process of modifying urokinase inhibitors based on (benzothiazol-3-yl)guanidine was developed. A new urokinase inhibitor (5-brom-benzothiazol-3-yl)guanidine, that can be effective for regulation of vascular remodeling and tumor angiogenesis, was created.

[Mechanisms of Vascular Remodeling Following Arterial Injury].

Arterial remodeling is the process of adaptation of the vessel comprising multiple structural and functional alterations of the vascular wall that occur in disease, trauma or aging. Arterial remodeling is accelerated in conditions that adversely affect the structural and functional balance of the vascular system, such as hypertension, atherosclerosis, kidney disease, inflammatory diseases, genetic abnormalities, and mechanical damage. Pathological changes in the vascular wall lead to organ damage and, ultimately, death. The aim of this paper is to review the various factors and complex mechanisms, which underlie negative arterial remodeling after mechanical injury and data indicating on the key role of the urokinase plasminogen activator in this process.

Urokinase stimulates production of matrix metalloproteinase-9 in fibroblasts with involvement of reactive oxygen species.

In cultured fibroblasts, urokinase stimulated expression of MMP-9 and generation of ROS, while antioxidant ebselen abolished the stimulating effect of urokinase on MMP-9 expression. sTNF-α produced similar and more pronounced stimulating effect. The data showed that urokinase could regulate MMP-9 expression via ROS generation in fibroblasts, which can play an important role in stimulation of their migration and development of constrictor (negative) vascular remodeling due to thickening of the adventitia.

Proteolytically inactive recombinant forms of urokinase suppress migration of endothelial cells.

Proteolytically inactive recombinant forms of urokinase (uPAHQ and amino-terminal fragment) inhibit spontaneous migration of endothelial cells; amino-terminal fragment also suppresses angiogenesis stimulated by basic fibroblast growth factor in vitro. These findings suggest the possibility of using synthesized proteolytically inactive recombinant forms of urokinase for the regulation of endothelial cell migration and suppression of neoangiogenesis.

[The evaluation and comparative characteristic of detection of clone of paroxysmal nocturnal hemoglobinuria on reticulocytes using the technique of flow cytometry].

The paroxysmal nocturnal hemoglobinuria is a rare clonal disease characterized by somatic mutation of gene PIG-A at the level of stem hematopoietic cell. This process results in disorder of synthesis of glycosil phosphatidyl innozitol (GPI) anchor fixing numerous molecules on membrane of blood cells which protect blood cells from impact of complement. The international society of clinical cytometry (2010) proposed the guidelines of detection of clone of paroxysmal nocturnal hemoglobinuria among erythrocytes, granulocytes and monocytes. The original technique is proposed to evaluate the clone of paroxysmal nocturnal hemoglobinuria in reticulocyte population of blood using method of flow cytofluorometry. The sampling of 160 samples of blood of patients with clinical symptoms of paroxysmal nocturnal hemoglobinuria and anemia was analyzed. Two modes of gatedrawing were applied--using monoclonal antibodies to CD71 (receptor to transferrin) and reagent BD ReticCount. The high correlation was established between size of reticulocytic clone of paroxysmal nocturnal hemoglobinuria evaluated by CD71 and size of granulocytic and monocytic clone of paroxysmal nocturnal hemoglobinuria. The developed panel (CD71/CD235a/CD59) can be applied for screening and monitoring of paroxysmal nocturnal hemoglobinuria.

[The standardization of diagnostic of paroxysmal night hemoglobinuria cytometry].

The flow cytometry becomes a more and more largely applied technique. However, the sufficient novelty of technique has no worked-out standards of diagnostic of many diseases. The lacking of external control of quality promotes development of large variety of approaches to diagnostic of diseases and impossibility to compare the study results from different laboratories. The paroxysmal night hemoglobinuria is an acquired clonal disease characterized by proliferation of stem cells with partial or total loss of expression of glykosylphosphosphatidyl inositol anchor needed to conjugate a number of surface proteins. The flow cytometry is a basic technique of detection and monitoring of clone of paroxysmal night hemoglobinuria. The article presents the results of paroxysmal night hemoglobinuria testing of 8 patients in 6 independent laboratories using flow cytometry by standard protocol recommended by the International society of clinical cytometrists (ICCS).

[Plasminogen activators and matrix metalloproteinases during arterial remodeling].

To evaluate the role and interaction of plasminogen activators and matrix metalloproteinases (MMPs) in arterial remodeling in vivo we compared effects of recombinant urokinase- (uPA) and tissue-type (tPA) plasminogen activators on vessel morphology, cell proliferation, inflammatory reaction and MMPs expression in arterial wall after experimental balloon angioplasty. We observed that the periadventitial application of uPA to the injured artery in pluronic gel stimulated neointima formation and inward arterial remodeling as well as cell proliferation and inflammatory leukocytes recruitment. In contrast, tPA attenuated neointima growth, contributed to outward arterial remodeling and did not affect significantly leukocytes recruitment in injured arterial wall. Perivascular uPA increased the content and activity of MMPs, while tPA did not induce such changes. In mouse model of vascular remodeling based on partial ligation of the carotid the content of uPA correlated with neointima growth, tPA content correlated with outward arterial remodeling. Our experiments suggest that plasminogen activators represent specific functional target for attenuating unfavorable inward arterial remodeling.

Urokinase plasminogen activator enhances neointima growth and reduces lumen size in injured carotid arteries.

OBJECTIVES: Increases in urokinase plasminogen activator (uPA) have been reported in tissues undergoing remodelling, but its effects on the vessel intima formation are not known. We investigated its effects on carotid artery intima, media and lumen size, as well as smooth muscle cell (SMC) proliferation and migration in vivo. DESIGN AND METHODS: Carotid arteries of rats were distended with an inflated balloon catheter and uPA, or uPA-neutralizing antibodies were applied perivascularly in pluronic gel; control rats received vehicle. Carotid artery structure, cell migration and proliferation were assessed after 4 days by quantitative morphometry and immunohistochemistry. RESULTS: Four days after increasing vessel uPA, the intima/media ratio was double compared to that in control rats (both P < 0.05). The size of the lumen reduced by 75%, compared to the vehicle-treated vessels (P < 0.05). The elevation in uPA also increased SMC numbers in the intima and media, compared to the vehicle-treated vessels (both P < 0.05). Antibody neutralizing endogenous uPA attenuated the growth responses in the distended arteries, reduced neointimal SMC numbers by approximately 50% and prevented much of the reduction in lumen size. CONCLUSIONS: Thus, local increases in uPA in distended, injured arteries augment SMC migratory and proliferative responses, leading to increases in the thickness of the carotid artery intima and media and a reduction in lumen size; effects at least partially attributable to its proteolytic properties.

[Expression of urokinase plasminogen activator, its receptor and plasminogen activator inhibitor type 1 in different types of atherosclerotic lesion in human aorta]. / Ekspressiia urokinazy, ee retseptora i ingibitora aktivatorov plazminogena 1-go tipa v stenke aorty cheloveka pri raznykh tipakh ateroskleroticheskogo porazheniia.

The role of plasminogen activators in the regulation of key processes of atherosclerosis progression stays unclear. The aim of this study was to evaluate the expression of urokinase plasminogen activator (uPA), its receptor (uPAR) and the plasminogen activator inhibitor type 1 (PAI-1) in human aorta, and to balance them with the stage of atherosclerotic lesion. We have shown that uPA and uPAR in normal aorta are mostly expressed by intimal smooth muscle cells. The expression of these proteins was up-regulated in diseased aorta compared to normal artery. The most part of cells in both fatty streak and fibro-fatty lesion were monocytes/macrophages, and about 60% of these cells expressed uPA and its receptor. PAI-1 was mostly localized on the lumonal part of the aorta and in the extracellular matrix of the intima. We observed a moderate increase of PAI-1 expression in atherosclerotic lesion. Thus, our data indicate participation of plasminogen system in atherogenesis.

Application of molecular modeling to urokinase inhibitors development.

Urokinase-type plasminogen activator (uPA) plays an important role in the regulation of diverse physiologic and pathologic processes. Experimental research has shown that elevated uPA expression is associated with cancer progression, metastasis, and shortened survival in patients, whereas suppression of proteolytic activity of uPA leads to evident decrease of metastasis. Therefore, uPA has been considered as a promising molecular target for development of anticancer drugs. The present study sets out to develop the new selective uPA inhibitors using computer-aided structural based drug design methods. Investigation involves the following stages: computer modeling of the protein active site, development and validation of computer molecular modeling methods: docking (SOL program), postprocessing (DISCORE program), direct generalized docking (FLM program), and the application of the quantum chemical calculations (MOPAC package), search of uPA inhibitors among molecules from databases of ready-made compounds to find new uPA inhibitors, and design of new chemical structures and their optimization and experimental examination. On the basis of known uPA inhibitors and modeling results, 18 new compounds have been designed, calculated using programs mentioned above, synthesized, and tested in vitro. Eight of them display inhibitory activity and two of them display activity about 10 µM.

[Regulation of growth and remodeling of blood vessels: the unique role of urokinase].

Urokinase-plasminogen activator (uPA) is a multifunctional fibrinolytic protein activating growth factors, inducing proteolytic cascades, modulating cytokines, regulating receptor shedding, cellular phenotypic modulation and protein expression. These mechanisms underlie the ability of uPA to stimulate the key processes of vascular remodelling, atherosclerosis progression, restenosis and angiogenesis, -- cell migration and proliferation. We summarized data received by us and others concerning the role of uPA in blood vessel remodelling and growth. At the present stage, the uPA may be considered as a perspective target for influences directed on both the prevention of negative arterial remodelling and restenosis as well as the stimulation of vessel growth at ischemic diseases.

Urokinase stimulates inflammatory response in damaged vascular wall during in vivo arterial remodeling.

Perivascular application of urokinase to the ballooned artery promoted the growth of neointima and constrictive remodeling of the vessel and stimulated the inflammatory response in the damaged vascular wall in vivo. Recombinant tissue plasminogen activator did not induce these changes. Our results indicate that urokinase is involved in the regulation of the inflammatory response during in vivo remodeling of the damaged vascular wall.

Urokinase induces ROS production in vascular smooth muscle cells.

Urokinase stimulates the production of superoxide radical in cultured aortal smooth muscle cells simultaneously with activation of the expression of NAD(F)H-oxidases nox1, nox4, and phox22. Antioxidant ebselen abolishes the stimulating effect of urokinase on smooth muscle cell proliferation. The data showed that urokinase can potentiate oxidative stress in the arterial wall and can play an important role in the development of adverse arterial remodeling.

Urokinase increases the content and activity of matrix metalloproteinases 2 and 9 during in vivo constrictive arterial remodeling.

Perivascular application of urokinase to ballooned artery stimulating the formation of neointima and constrictive arterial remodeling increased the content and activity of matrix metalloproteinases 2 and 9 in vivo. Application of recombinant tissue plasminogen produced no such changes. It was demonstrated that urokinase stimulates expression of matrix metalloproteinases in vivo.

Plasminogen activators in vascular remodeling and angiogenesis.

This review considers cellular and molecular mechanisms of the involvement of plasminogen activators in extracellular proteolysis and cell migration and proliferation. The role of plasminogen activators in vascular remodeling in atherosclerosis, restenosis, and angiogenesis is discussed.

Urokinase stimulates differentiation of fibroblasts into myofibroblasts and their proliferation in damaged adventitia.

Urokinase expression in the adventitia of rat common carotid artery increased on the 4th day after periadventitial damage. Periadventitial application of recombinant urokinase increased the area of the adventitia and the content of contractile and proliferating cells, while proteolytically inactive recombinant urokinase was ineffective.

[Expression of urokinase and its receptor correlate with proliferation of smooth muscle cell in injured arteries]. / Ekspressia urokinazy i ee retseptora korreliruet s proliferatsiei gladkomyshechnykh kletok v povrezhdennykh arteriiakh.

Using the immunohistochemistry and a standard reverse transcriptase-polymerase chain reaction assay optimized to estimate the mRNA levels, we observed a 2-fold increased uPA expression by the SMCs in injured vessels as compared with uninjured vessels. The uPA elevation occurred within 6 hours from the injury and persisted for 96 hours after the injury. The uPAR expression was also elevated after an injury although it occurred slower and more gradually. The data obtained suggest that the uPA is capable of contributing to both the SMC migration, replication and accumulation in the neointima early after balloon catheter injury of the carotid artery in rats.

[Urokinase stimulates whereas tissue plasminogen activator attenuates blood vessel stenosis]. / Urokinaza stimuliruet, a tkanevoi aktivator plazminogena podavliaet razvitie stenoza krovenosnykh sosudov.

Periadventitial application of the urokinase-plasminogen activator (uPA) in pluronic gel to an injured artery stimulated the neointima and neoadventitia formation as well as cell migration and proliferation in vivo. In contrast, the tissue-type plasminogen activator (tPA) reduced the number of neointimal smooth muscle cells and neointimal area and attenuated the lumen stenosis after a balloon catheter injury of the rat carotid artery. This ability to stimulate the neointima and neoadbentitia formation was found to be quite specific for the uPA. The findings suggest that this uPA property provides a specific functional target for attenuating growth of the damage.