PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but its role in PAH is unsettled. Here, we report that: (1) PAI-1 is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared to controls; (2) PAI-1-/- mice spontaneously develop pulmonary vascular remodeling associated with up-regulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and (3) pharmacological inhibition of uPA in human PAH PASMCs suppresses pro-proliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that down-regulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent up-regulation of mTOR and TGF-b signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.

Packaging of supplemented urokinase into alpha granules of in vitro-grown megakaryocytes for targeted nascent clot lysis.

ABSTRACT: Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.

Packaging of supplemented urokinase into naked alpha-granules of in vitro -grown megakaryocytes for targeted therapeutic delivery.

Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human platelets in vitro from CD34 + hematopoietic cell-derived megakaryocytes. CD34 + -megakaryocytes internalize and store in α-granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34 + -\megakaryocytes was mediated in part via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF, in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWF R1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWF R1326H generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet α-granules from in vitro- generated megakaryocytes. Key points: Unlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its α-granules.uPA uptake involves LRP1 and αIIbß3 receptors and is functionally available from activated platelets.

PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

Seasonal Changes in the Antioxidant Activity and Biochemical Parameters of Goat Milk.

Goats are ubiquitous, including in hot and dry regions, while also being very sensitive to climate fluctuations, expressed in temperature differences. This affects their productivity and milk quality. Adaptation to heat requires high energy costs, affects "neurohumoral" regulation and is accompanied by oxidative stress with the increased production of free radicals. The aim was to study the main biochemical parameters of goat milk and its antioxidant activity depending on the season of the year. Sampling was carried out in April, June, August and October. Analysis of the biochemical components and antioxidant activity of goat milk was performed using modern analytical systems. From spring to autumn, the mass fraction of true or crude proteins in goat milk increased by 14.6-63.7% or by 12.3-52.1%, and the mass fraction of caseins also increased by 13.6-60.6%. For vitamin C level and the total amount of water-soluble antioxidants, a pronounced gradual decrease from spring to autumn was observed. In the summer period, a small increase in the carotene level in milk (by 3.0-6.1% compared to April) was established. Vitamin A content increased by 86.5% (June) or by 70.3% (October) compared to April. Thus, the numerous significant changes in the major parameters of goat's milk depending on the season were revealed.

Relationships between the Content of Micro- and Macroelements in Animal Samples and Diseases of Different Etiologies.

Many of the micro- and macro-elements (MMEs) required by the body are found in environmental objects in concentrations different from their original concentration that can lead to dangerous animal diseases ("microelementoses"). The aim was to study the features of MME (accumulating in wild and exotic animals) in connection with particular diseases. The work using 67 mammal species from four Russian zoological institutions was completed in 2022. Studies of 820 cleaned and defatted samples (hair, fur, etc.) after "wet-acid-ashing" on an electric stove and in a muffle furnace were performed using a Kvant-2A atomic absorption spectrometer. The content of zinc, copper, iron, cadmium, lead, and arsenic was assessed. The level of MME accumulation in the animal body contributes not only to the MME status and the development of various concomitant diseases, but the condition itself can occur by intake of a number of micronutrients and/or drugs. Particular correlations between the accumulation of Zn and skin, oncological diseases, Cu-musculoskeletal, cardiovascular diseases, Fe-oncological diseases, Pb-metabolic, nervous, oncological diseases, and Cd-cardiovascular diseases were established. Therefore, monitoring of the MME status of the organism must be carried out regularly (optimally once every 6 months).

Modulation of ultralarge immune complexes in heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a serious thrombotic disorder caused by ultralarge immune complexes (ULICs) containing platelet factor 4 (PF4) and heparin that form the HIT antigen, together with a subset of anti-PF4 antibodies. ULICs initiate prothrombotic responses by engaging Fcγ receptors on platelets, neutrophils, and monocytes. Contemporary anti-thrombotic therapy for HIT is neither entirely safe nor entirely successful and acts downstream of ULIC formation and Fcγ receptor-initiated generation of thrombin. OBJECTIVES: To determine whether HIT antigen and ULIC formation and stability could be modified favorably by inhibiting PF4-heparin interactions with fondaparinux, together with blocking formation of PF4 tetramers using a humanized monoclonal anti-PF4 antibody (hRTO). METHODS: Results: The combination of fondaparinux and hRTO inhibited HIT antigen formation, promoted antigen dissociation, inhibited ULIC formation, and promoted ULIC disassembly at concentrations below the effective concentration of either alone and blocked Fcγ receptor-dependent induction of factor Xa activity by monocytic THP1 cells and activation of human platelets in whole blood. Combined with hRTO, fondaparinux inhibited HIT antigen and immune complex formation and activation through Fcγ receptors at concentrations at or below those used clinically to inhibit FXa coagulant activity. CONCLUSIONS: HIT antigen and immune complexes are dynamic and amenable to modulation. Fondaparinux can be converted from an anticoagulant that acts at a downstream amplification step into a rationale, disease-specific intervention that blocks ULIC formation. Interventions that prevent ULIC formation and stability might increase the efficacy, permit use of lower doses, shorten the duration of antithrombotic therapy, and help prevent this serious thrombotic disorder.

NiB-CrC Coatings Prepared by Magnetron Sputtering Using Composite Ceramic NiCr-BC Target Produced by Detonation Spray Coating.

A metal-ceramic composite target for magnetron sputtering was fabricated for the first time by a robotic complex for the detonation spraying of coatings equipped with a multi-chamber detonation accelerator. A mixture of metal and ceramic NiCr/B4C powders was sprayed onto the copper base of the cylindrical composite target cathode. The study of the structure of a metal-ceramic composite coating target using scanning electron microscopy showed that the coating material is dense without visible pores; the elemental composition is evenly distributed in the material. The study of the cathode sputtering area after deposition in the DC mode showed that there are uniform traces of annular erosion on the target surface. The obtained cathode target with an NiCr-70B4C coating was used to deposit the NiB-Cr7C3 coating on flat specimens of 65G steel using equipment for magnetron sputtering UNICOAT 200. The coating was applied in the Direct Current mode. A dense NiB-Cr7C3 coating with a thickness of 2 µm was obtained. The NiB-Cr7C3 coating has a quasi-amorphous structure. The microstructures and concentration of oxygen and carbon impurities throughout the entire thickness of the coating were investigated by means of transmission electron microscopy. The results of the study show that the coatings have a nanocrystalline multi-phase structure. The microhardness of the NiB-Cr7C3 coating reached 10 GPa, and the adhesion fracture load exceeded 16 N. The results will open up new prospects for the further elaboration of technology for obtaining original composite cathodes for magnetron sputtering using detonation spraying of coatings.

Metabolic Profile of Sow Blood Serum after Weaning.

The aim of our research was to determine the content of protein, carbohydrate, lipid, and mineral metabolites, as well as an antioxidant status of the sow's blood after weaning and to calculate the correlation between these parameters. The experiment was carried out on twenty clinically healthy crossbred sows (Yorkshire × Landrace). Twenty sows were allocated to one of two groups: (1) 1 day after weaning (group 1, n = 10) and (2) 8 days after weaning (group 2, n = 10). The basis of the sow diet was SK-1 compound feed, balanced in terms of nutrients and energy in accordance with modern standards and the recommended feeding regimen. Sows blood samples were taken and analyzed for the metabolites of nitrogenous, carbohydrate-lipid, and mineral metabolism and indicators of antioxidant status. The results showed that, in group 2, the total protein content was 89.07 g/l, which is 10.2% higher than that in group 1 (p < 0.05); it was mainly achieved due to the globulin fraction. The urea increased by 19.1% (p < 0.05), but the concentrations of magnesium and chlorides decreased by 20.2% (p < 0.01) and 5.43% (p < 0.05), as well as the alkaline phosphatase and ALT activities decreased by 42.5% (p < 0.05). Strong positive correlations of the ceruloplasmin with total protein (0.672) and very strong with globulins (0.780) were observed. There was a strong negative correlation between the AST activity and the TBA-AP content, as well as the values of phospholipids and TAWSA. There are moderate negative correlations of the TBA-AP with magnesium, TAWSA and ALT activity, and moderate positive correlations of the TBA-AP with total protein, albumin, triglycerides, and cholesterol. The revealed tendencies and dependencies will serve as the theoretical basis for the development of practical methods for regulating the level of free-radical reactions.

Correlations between the Major Amino Acids and Biochemical Blood Parameters of Pigs at Controlled Fattening Duration.

Analytical control of protein and amino acid (AA) contents of animal tissues is an important problem in the fundamental and applied aspects. The aims of the work were the following: to measure the pig blood AAs; and to establish the correlations between AAs and biochemical parameters in dependence on the pig fattening duration. All 80 animals were divided onto 4 animal groups: 65, 72, 82, and 90 fattening days. The correlations between AAs and the total protein or its fractions (TP&F), nitrogen metabolites, carbohydrates, lipids, some enzymes in the pig blood for each of these animal groups obtained for the first time. The authors established the following total amounts of correlation coefficients (with reasonable p-values) in each of the group separately: group 1, 1* (p < 0.05); group 2, 0; group 3, 28* (p < 0.05) and 9** (p < 0.01); group 4, 28* (p < 0.05) and 25** (p < 0.01). Thus, about 82−90 days (groups 3 and 4) can be the optimal for the pig fattening, based on the correlation analysis for the numerous data of major AA and biochemical parameters of pig blood. These results can be useful for animal health monitoring and husbandry.

Correlations between the Total Antioxidant Activity and Biochemical Parameters of Cow Milk Depending on the Number of Somatic Cells.

The aim of the work was to study the correlations between the total amount of water-soluble antioxidants (TAWSA) and biochemical parameters (BC) of cow milk depending on the somatic cell count (SCC). The BC and TAWSA values of cow milk were measured by spectroscopic and amperometric methods, respectively. The milk samples from the black-and-white cows (Moscow region) were divided according to SCС values: (1) ≤200, (2) 200-499, (3) 500-999, and (4) ≥1000 thousand units/mL. The average TAWSA values for groups 1, 2, 3, and 4 (33, 15, 13, and 12 milk samples) were the following: 15.95 ± 0.74, 14.45 ± 0.84, 16.04 ± 0.63, and 14.58 ± 1.18. The correlations between TAWSA and BC (group 1) were the following: total fat percentage (TFP) -0.305; true protein percentage (TP1) -0.197; total nitrogen percentage (TN2) -0.210; lactose -0.156; solids-not-fat (SNF) -0.276; total dry matter (TDM) -0.399; freezing point (FP) -0.112; pH -0.114; somatic cell count (SCC) - (-0,052). The correlations between TAWSA and BC (group 2) were the following: TFP -0.332; TP1 -0.296; TN2 -0.303; lactose - (-0.308); SNF -0.159; TDM -0.391; FP -0.226; pH - (-0.211); SCC -0.193. The correlations between TAWSA and BC (group 3) were the following: TFP - (-0.352); TP1 - (-0.411); TN2 - (-0.401); lactose - (-0.166); SNF - (-0.462); TDM - (-0.504); FP - (-0.766); pH - (-0.047); SCC - (-0.698). The correlations between TAWSA and BC (group 4) were the following: TFP -0.159; TP1 -0.046; TN2 - 0.077; lactose - (-0.317); SNF - (-0.237); TDM -0.058; FP - (-0.036); pH - (-0.477); SCC - (-0.072). These data are important in assessing the physiological-biochemical status and state of the antioxidant defense system of cows' organism.

Effectiveness of bortezomib and temozolomide for eradication of recurrent human glioblastoma cells, resistant to radiation.

BACKGROUND: Glioblastoma multiforme (GBM) is a primary human brain tumor with the highest mortality rate. The prognosis for such patients is unfavorable, since the tumor is highly resistant to treatment, and the median survival of patients is 13 months. Chemotherapy might extend patients' life, but a tumor, that reappears after chemoradiotherapy, is resistant to temozolomide (TMZ). Using postgenome technologies in clinical practice might have a positive effect on the treatment of a recurrent GBM. METHODS: T98G cells of human GBM have been used. Radiation treatment was performed with Rokus-M gamma-therapeutic system, using 60Сo as a source of radionuclide emissions. High-performance liquid chromatography-mass spectrometry was used for proteome analysis. Mass spectrometry data were processed with MaxQuant (version 1.6.1.0) and Perseus (version 1.6.1) software, Max Planck Institute of Biochemistry (Germany). Biological processes, molecular functions, cells locations and protein pathways were annotated with a help of PubMed, PANTHER, Gene Ontology and KEGG and STRING v10 databases. Pharmaceutical testing was performed in vitro with a panel of traditional chemotherapeutic agents. RESULTS: GBM cells proliferation speed is inversely proportional to the irradiation dose and recedes when the dosage is increased, as expected. Synthesis of ERC1, NARG1L, PLCD3, ROCK2, SARNP, TMSB4X and YTHDF2 in GBM cells, treated with 60Gy of radiation, shows more than a fourfold increase, while the synthesis level of PSMA2, PSMA3, PSMA4, PSMB2, PSMB3, PSMB7, PSMC3, PSMD1, PSMD3 proteins increases significantly. Traditional chemotherapeutic agents are not very effective against cancer cells of the recurrent GBM. Combination of TMZ and CCNU with a proteasome inhibitor-bortezomib-significantly increases their ability to eradicate cells of a radioresistant GBM. CONCLUSIONS: Bortezomib and temozolomide effectively destroy cells of a radioresistant recurrent human glioblastoma; proteome mapping of the recurrent GBM cancer cells allows to identify new targets for therapy to improve the treatment results.

Correlations between Antioxidant and Biochemical Parameters of Blood Serum of Duroc Breed Pigs.

Correlations between the major biochemical (BC) and antioxidant (TAWSA) parameters of pigs' blood are necessary to study in order to assess physiological-biochemical status (PhBS), animal health, production, etc. Blood samples were obtained from Duroc breed boars (n = 77), divided into groups 1 (n = 25), 2 (n = 40) and 3 (n = 12), which were fattened for 65, 72 and 100 days, respectively. Significant positive and negative correlations were found between TAWSA and BC parameters of pigs' blood for group 3: very high in the case of total protein (TP) (-0.75) and aspartate aminotransferase (AST) (-0.79); high in the case of cholesterol (-0.72), glucose (0.66), alkaline phosphatase (0.66), calcium ions (-0.60) and globulins (0.53); moderate in the case of albumins (-0.36), triglycerides (-0.35), magnesium (-0.32) and phosphorus (-0.27). The same was found for group 2: high in the case of TP (0.51); moderate in the case of globulins (0.48), cholesterol (0.33) and phosphates (0.25). The only moderate correlation was found for group 1: magnesium (-0.48), glucose (0.36) and calcium (-0.25). This tendency indicated the stabilization of pig PhBS during growth and fattening, which can be useful for understanding the PhBS and antioxidant features of pigs, the factors of their nutrition, maintenance, etc.

Biochemical parameters, dynamic tensiometry and circulating nucleic acids for cattle blood analysis: a review.

The animal's blood is the most complicated and important biological liquid for veterinary medicine. In addition to standard methods that are always in use, recent technologies such as dynamic tensiometry (DT) of blood serum and PCR analysis of particular markers are in progress. The standard and modern biochemical tests are commonly used for general screening and, finally, complete diagnosis of animal health. Interpretation of major biochemical parameters is similar across animal species, but there are a few peculiarities in each case, especially well-known for cattle. The following directions are discussed here: hematological indicators; "total protein" and its fractions; some enzymes; major low-molecular metabolites (glucose, lipids, bilirubin, etc.); cations and anions. As example, the numerous correlations between DT data and biochemical parameters of cattle serum have been obtained and discussed. Changes in the cell-free nucleic acids (cfDNA) circulating in the blood have been studied and analyzed in a variety of conditions; for example, pregnancy, infectious and chronic diseases, and cancer. CfDNA can easily be detected using standard molecular biological techniques like DNA amplification and next-generation sequencing. The application of digital PCR even allows exact quantification of copy number variations which are for example important in prenatal diagnosis of chromosomal aberrations.

Proteins of Wnt signaling pathway in cancer stem cells of human glioblastoma.

RATIONALE: Glioblastoma multiforme (GBM) is the most aggressive primary glial brain tumor. The prognosis for GBM patients is not favorable, with the median survival time being 15 months. Its treatment resistance is associated with GBM cell population having cancer stem cells (CSCs). Wnt/ß-catenin signaling pathway is a strategically important molecular mechanism, providing proliferation of stem cells of all types. This study compares the expression levels of signaling pathway proteins in CD133(+) CSCs and CD133(-) differentiated glioblastoma cells (DGCs). MATERIALS AND METHODS: the present study used U-87MG cells of human glioblastoma, the material was tested for mycoplasma contamination. High-performance liquid chromatography (HPLC) mass spectrometry was used for proteome analysis. Biological and molecular functions, signaling pathways and protein-protein interactions were analyzed using free-access databases: PubMed, PANTHER, Gene Ontology, Swiss-Prot and KEGG. Protein-protein interactions (PPIs) were analyzed using the STRING database (version 10). RESULTS: There were identified 589 proteins with significantly changed expression in CD133+ CSCs, as compared with CD133-DGCs (P<0.05). Bioinformatics analysis allowed to attribute 134 differentially expressed proteins to 16 signaling pathways. A significant increase in expression of eight Wnt signaling pathway proteins (APC, CSNK1E, CSNK1A, CSNK2A2, CSNK2B, CTNNB1, DVL1, RUVBL) was detected, as well as four proteins of the non-canonical Wnt pathway-RHOA, ROCK2, RAC2, DAAM1. Special attention should be paid to ß-catenin (CTNNB1) with more than 13.98-fold increase of expression in CSCs and Disheveled-associated activator of morphogenesis 1 (DAAM1) with 6.15-fold higher upregulation level. CONCLUSION: proteins of Wnt/ß-catenin signaling cascade are a prospective target for regulating CSCs activity.

Transforming growth factor-ß mimics the key proteome properties of CD133- differentiated and CD133+ cancer stem cells in glioblastoma.

Glioblastoma multiforme is the most aggressive type of primary brain tumor in humans. Its invasive growth is associated with cluster of differentiation (CD)133 cancer stem cells (CSCs) and CD133- differentiated glioblastoma cells (DGCs) with aggressive phenotype, which are developed under the influence of transforming growth factor (TGF)-ß. The present study aimed to compare the proteomes of CD133 CSCs and CD133- DGCs stimulated by TGF-ß, as well as the expression levels of the main proteins responsible for activating the signaling pathway of receptor interactions with the extracellular matrix (ECM). The U87MG GBM cell line was used in this study. CSCs were extracted from gliomaspheres through magnetic-activated cell sorting based on the expression of CD133 (CD133); CD133- DCGs served as a control. CD133- DGCs of the U87-MG cell line were treated with 10ng/mL TGF-ß1, and cell proliferation and migration were analyzed via real-time quantitative microscopy. High-performance liquid chromatography mass spectrometry was used for proteome analysis. The results revealed 589 proteins with significantly changes in expression among CD133 CSCs compared with those in CD133- DGCs (P<0.05). Bioinformatics analysis allowed to attribute 134 differentially expressed proteins to 15 signaling pathways; among these proteins, 14 were involved in signaling cascades associated with the interaction between CSCs and the ECM, and were upregulated >twofold, while four proteins activated this signaling cascade. TGF-ß-stimulation increased the mobility, suppressed the proliferation and transformed the proteome profile of CD133- DGCs. Were identified 13 key proteins that activate the signaling pathway of receptor interaction with the ECM and three proteins activating this signaling pathway in CD133- DGCs which had the same values as those of CD133 CSCs. In conclusion, TGF-ß increased the expression of proteins that activate the signaling pathway of receptor interaction with the ECM in CD133- DGCs to the level of those in CD133 CSCs.

Pro-inflammatory modification of cancer cells microsurroundings increases the survival rates for rats with low differentiated malignant glioma of brain.

RATIONALE: Glioblastoma multiforme (GBM) is one of the most aggressive human brain tumors. The prognosis is unfavorable with a median survival of 15 months. GBM aggressive nature is associated with a special phenotype of cancer cells that develops because of the transforming growth factor ß (TGF-ß). The study was aimed at providing experimental justification in vivo of a possibility to suppress TGF-ß production in a tumor via pro-inflammatory modification of cancer cell microenvironment, using CD45+ mononuclear cells of the red bone marrow. MATERIALS AND METHODS: The experiment used animals with transplanted C6 glioma. The animals were divided into 4 groups: (I) control (N=60); (II) group of rats (N=30) that received granulocyte colony-stimulating factor (G-CSF) to recruit CD45+ bone marrow mononuclear cells into their systemic circulation (G-CSF group); (III) group of rats (N=30) that received pro-inflammatory therapy to trigger systemic inflammatory reaction by injecting bacterial lipopolysaccharides (LPS) and interferon-γ (IFNγ); (IV) rats (N=30), stimulated with G-CSF, followed by pro-inflammatory therapy. Stereotaxic modeling of a brain tumor in experimental animals, as well as a combination of morphological, immunocytochemical analyses and immunosorbent assay were used. RESULTS: TGF-ß1 production in the tumor tissue resulted being inversely proportional to the intensity of proliferation processes and directly proportional to the size of necrosis areas, peaking on the 28th day of the experiment. Stimulation of experimental animals with G-CSF recruits CD45+ mononuclear stem and progenitor cells into the systemic circulation of experimental animals with C6 glioma, accompanied by intensification of microglial proliferation in the tumor and infiltration of the tumor tissue with microglial cells. Pro-inflammatory therapy against G-CSF stimulation results in polarization of microglia/macrophages population together with intensified antigen presentation, lower production of TGF-ß and IL10, increased synthesis of pro-inflammatory cytokines TNFα and IL1 in the tumor lesion and adjacent brain matter, remodeling of tumor matrix and higher survival rates for the experimental animals. CONCLUSIONS: Pro-inflammatory inflammatory modification of cancer cell microenvironment suppresses TGFß production in a tumor and increases survival rates of the rats with transplanted poorly differentiated malignant brain glioma.

FcRn augments induction of tissue factor activity by IgG-containing immune complexes.

Thromboembolism complicates disorders caused by immunoglobulin G (IgG)-containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), ß-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.

Biochemical aspects of lipase immobilization at polysaccharides for biotechnology.

The design of immobilized enzyme preparations is an important and relevant area of modern sciences and technologies. Immobilization of enzymes from animal sources (component I) on natural carriers (component II) increases the system stability by protecting the active site of the enzyme from deactivation; facilitates the separation and accelerates the recovery of the enzyme. This makes reuse possible and provides a significant reduction in operating costs. Hydrolytic enzymes (such as lipases) and polysaccharides (such as chitosan) are the most promising of such pairs of components. The main attention here is devoted to the discussion on lipase immobilization on polysaccharide (mainly - chitin and chitosan). Based on the analysis of the available literature, the most adequate method is the immobilization of lipase from porcine pancreas (LPP) on polysaccharide particles (such as chitin or chitosan) pre-treated with ultrasound (to increase the particle surface area) and glutaraldehyde (for particle activation) that shows reasonably high LPP activity and stability. In order to increase further the activity of the lipase, some authors proposed to incorporate a spacer in the form of 1,3-diaminopropane (or 1,3-diaminobutane) prior to activation of the surface of the chitosan particles. In particular cases, the use of chitin (instead of chitosan) may be an alternative solution for biotechnological applications. Recently the idea of constructing "supramolecular enzyme systems" realized in the so-called "coimmobilized multienzymatic systems" strategy. The most fascinating example is the combined assay of a mixture of native LPP, glycerol kinase (from Cellulomonas) and glycerol-3-phosphate oxidase (from Aerococcus viridans) linked by glutaraldehyde to chitosan (as shell for inorganic nanoparticle core). This material was placed on a Pt-electrode as biosensor and was successfully applied for amperometric determination of the triglyceride level in the serum of healthy and diseased person. Thus, the whole innovative research-production sequence is described by Aggarwal V. and Pundir C.S.: from simple components to advanced material and further biomedical application. Thus, the following approach of lipase immobilization appears the most promising for future applications: a few types of lipases or the combination of LPP with some other enzymes immobilized simultaneously on multifunctional carriers (as nanohybrids of inorganic core and polysaccharide shell).

Blood clot contraction differentially modulates internal and external fibrinolysis.

Essentials Clot contraction influences the rate of fibrinolysis in vitro. Internal fibrinolysis is enhanced ∼2-fold in contracted vs. uncontracted blood clots. External fibrinolysis is ∼4-fold slower in contracted vs. uncontracted blood clots. Contraction can modulate lytic resistance and potentially the clinical outcome of thrombosis. SUMMARY: Background Fibrinolysis involves dissolution of polymeric fibrin networks that is required to restore blood flow through vessels obstructed by thrombi. The efficiency of lysis depends in part on the susceptibility of fibrin to enzymatic digestion, which is governed by the structure and spatial organization of fibrin fibers. How platelet-driven clot contraction affects the efficacy of fibrinolysis has received relatively little study. Objective Here, we examined the effects of clot contraction on the rate of internal fibrinolysis emanating from within the clot to simulate (patho)physiological conditions and external fibrinolysis initiated from the clot exterior to simulate therapeutic thrombolysis. Methods Clot contraction was prevented by inhibiting platelet myosin IIa activity, actin polymerization or platelet-fibrin(ogen) binding. Internal fibrinolysis was measured by optical tracking of clot size. External fibrinolysis was determined by the release of radioactive fibrin degradation products. Results and Conclusions Clot contraction enhanced the rate of internal fibrinolysis ∼2-fold. In contrast, external fibrinolysis was ~4-fold slower in contracted clots. This dichotomy in the susceptibility of contracted and uncontracted clots to internal vs. external lysis suggests that the rate of lysis is dependent upon the interplay between accessibility of fibrin fibers to fibrinolytic agents, including clot permeability, and the spatial proximity of the fibrin fibers that modulate the effects of the fibrinolytic enzymes. Understanding how compaction of blood clots influences clot lysis might have important implications for prevention and treatment of thrombotic disorders.