Human Plasminogen Activator Research Articles

Biodistribution and Safety Studies of a Bicistronic Plasmid for Nerve Repair.

Gene therapy is one of the promising approaches for regenerative medicine. Local and long-term expression of essential growth factors allows to achieve the desired therapeutic effect. However, some aspects of prolonged usage of genetic constructs encoding growth factors, such as toxicity, mutagenicity, genotoxicity, and ability to disseminate from the injection site and mediate ectopic expression of therapeutic proteins, are poorly investigated. These aspects of gene therapy drugs' usage became the subject of this study. To study plasmid biodistribution, toxicity, mutagenicity, and genotoxicity, we used previously described bicistronic genetic construct encoding human brain-derived neurotrophic factor (hBDNF) and human urokinase plasminogen activator (huPA) for nerve repair. Biodistribution studies were conducted in mice: a course of intramuscular plasmid injections was followed by the study of the content of the plasmid (real-time polymerase chain reaction) and recombinant proteins (enzyme-linked immunosorbent assay) in murine organs and tissues. The study of the plasmid chronic toxicity was carried out on rats with registration of their weight dynamics, neurological status, emotional state, and blood test parameters. The mutagenicity of the plasmid was studied in an in vivo DNA comet test in mice. Plasmid genotoxicity was investigated in the model of somatic recombination in Drosophila females. We have shown that plasmids can disseminate from the injection site, but do not mediate ectopic expression of growth factors upon repeated intramuscular injections. The studied plasmid also does not reveal toxic, mutagenic, or genotoxic effects. During the toxicological study on rats, we have shown that daily injections of this genetic construct, despite its ability to disseminate from the injection site, do not affect the physical, cognitive, and emotional state of experimental animals. We have demonstrated the safety of the bicistronic plasmid, encoding hBDNF and huPA, upon its repeated administration. The properties of genetic constructs strongly depend on their sequence and delivery approach, which requires conducting of their safety studies in each specific case. Impact statement Gene therapy is one of the promising approaches for regenerative medicine. Local and long-term expression of essential growth factors allows to achieve the desired therapeutic effect. However, some aspects of prolonged usage of genetic constructs encoding growth factors, such as toxicity, mutagenicity, genotoxicity, and ability to disseminate from the injection site and mediate ectopic expression of therapeutic proteins, are poorly investigated. These aspects of gene therapy became the subject of this study. To our knowledge, this is a unique study that provides a thorough safety investigation of a bicistronic plasmid after its readministration.

Integrative identification of human serpin PAI-1 inhibitors from Dracaena dragon blood and molecular implications for inhibitor-induced PAI-1 allosterism.

Human plasminogen activator inhibitor-1 (PAI-1) is an important component of the coagulation system and has been recognized as a potential therapeutic target of diverse cardiovascular disorders. Previously, it was found that the extracts from the Chinese medicine Dracaena dragon blood have potent inhibitory activity against PAI-1, but it is unclear which constituents directly participate in the inhibition and how do they regulate PAI-1 at molecular level. Here, we describe an integrated strategy to identify the dragon blood's chemical constituents that can directly target PAI-1. With the strategy, five compounds 1-5 are hit as promising PAI-1 inhibitor candidates, from which three are measured to have high or moderate activity against PAI-1. In particular, the compound 3 is determined to exhibit the highest potency; this value is roughly comparable with the widely used PAI-1 inhibitor Tiplaxtinin. We further examine the molecular effect of compound 3 on PAI-1 conformation at structural level. It is supposed that small-molecule inhibitor regulates the reactive center loop (RCL) of PAI-1 through an allosterism, that is, binding of compound 3 to PAI-1 can allosterically stabilize RCL in latent form, thus promoting PAI-1 conformational conversion from metastable active form to the inactive latent form. Long-term atomistic simulations also demonstrate that removal of compound 3 can destabilize the structured β-stranded conformation of RCL in latent form, although the current simulations are still not sufficient to characterize the full conversion dynamics trajectory.

Effect of Obesity on Tissue Factor, Tissue Plasminogen Activator and Plasminogen Activator Inhibitor Among Obese People in Sapele Southern Nigeria

<i>Background/Objective</i>: Obesity is a medical condition in which adipose tissue accumulates to the extent of having adverse effect on certain procaogulant factors such as: Human Plasminogen Activator Inhibitor (PAI), tissue Plasminogen Activator (t-PA) and Tissue Factor (TF) of adults in Nigeria. This study therefore aims to evaluate the impacts of obesity on PAI, t-PA, and TF in obese adult in Sapele, South-South Nigeria. <i>Materials and Methods</i>: 312 adults with obesity and 103 normal adults (used as controls) with age between 18 and 65 years were enrolled for this study. 4.5mls of venous blood was collected into EDTA containers for procoagulant activity measurement. Human Plasminogen Activator Inhibitor (PAI-1), Human tissue Plasminogen Activator (t-PA), and Human Tissue Factor (TF) values were determined using Enzyme Linked Immunosorbent Assay (ELISA) methods. Collated data was analyzed using Student’s t-test, correlation and one-way ANOVA followed by LSD post-hoc test and results were expressed as mean±standard deviation. <i>Results</i>: The BMI (Body Mass Index) of obese subjects and control subjects were 36.82±0.55kg/m² and 20.43±0.29 kg/m² respectively at p<0.001. t-PA level of obese subject was 344.34±43.58pg/ml while control group had t-PA value of 192.46±10.65pg/ml. In addition, PAI of obese people was 367.24±27.49pg/ml and control subject had PAI value of 253.37±11.11pg/ml. Human Tissue Factor (TF) of obese subject and control subjects were 93.31±7.25pg/ml and 66.59±2.76pg/ml respectively at p<0.01. <i>Conclusion</i>: The values of Plasminogen Activator Inhibitor, tissue Plasminogen Activator, and Human Tissue Factor were higher in people with obesity when compare with non-obese control group in Sapele Southern Nigeria.

Adenovirus Biodistribution is Modified in Sensitive Animals Compared to Naïve Animals.

Pre-existing immune response against adenovirus could diminish transgene expression efficiency when Ad is employed in humans as gene therapy vector. We previously used Ad-hΔuPA (Recombinant adenovirus expressing human urokinase-type plasminogen activator) as antifibrotic gene therapy in cirrhosis models and demonstrated its effectiveness. As a further clinical approach, transient Cyclosporine A (CsA) immunosuppression was induced in cirrhotic animals to determine whether Ad-hΔuPA administration retained efficacy. Adenovirus sensitization was achieved by systemic administration of non-therapeutic Ad-βGal (Recombinant adenovirus expressing beta-galactosidase) after 4weeks of intraperitoneal carbon tetrachloride (CCl4) regimen. Cirrhosis induction continued up to 8weeks. At the end of CCl4 intoxication, immunosuppression was achieved with three CsA doses (40mg/kg) as follows: 24h before administration of Ad-hΔuPA, at the moment of Ad-hΔuPA injection and finally, 24h after Ad-hΔuPA inoculation. At 2 and 72h after Ad-hΔuPA injection, animals were sacrificed. Liver, spleen, lung, kidney, heart, brain, and testis were analyzed for Ad-biodistribution and transgene expression. In naïve animals, Ad-hΔuPA genomes prevailed in liver and spleen, while Ad-sensitized rats showed Ad genomes also in their kidney and heart. Cirrhosis and Ad preimmunization status notably diminished transgene liver expression compared to healthy livers. CsA immunosuppression in cirrhotic animals has no effect on Ad-hΔuPA biodistribution, but increments survival.

Dynamic Consequences of Inactivating the Catalytic Serine 195 to Methionine in the Human Urokinase-Type Plasminogen Activator (uPA)

Human urokinase-type plasminogen activator (uPA) is a serine protease that has implications in various health processes including anticoagulation, angiogenesis and cancer. The catalytic site of uPA is made by a histidine (H57), an aspartic acid (D102) and a serine (S195), that last one being responsible for the nucleophilic attack that starts the peptide bond hydrolysis. Studying the dynamics of uPA is key to develop strategies to control the activity of this serine protease, however, analyzing this protein by many biophysical techniques is very challenging because it self-degrades under the experimental conditions, such as high protein concentration or long incubation times. We hypothesized that mutating S195 to methionine would yield a stable catalytically inactive mutant that would biophysically behave similarly to the wildtype. Site-directed mutagenesis was used to create the mutant. The mutant protein was expressed in E. coli as inclusion bodies, refolded in vitro and purified by a series of chromatography steps including Ni-affinity chromatography, aprotinin-affinity chromatography and size exclusion chromatography. The dynamics in solution of the S195M mutant and the wildtype were studied by hydrogen-deuterium exchange mass spectrometry (HDXMS). There were no significant differences in most of the protein, except in the 140s loop. In this loop, there are three peptides that have less protection in the S195M mutant versus the wild type protease. This is significant due to the fact that the 140s loop is at the entrance to the active site of the protease. These results suggest that the S195M mutant is causing allostery in the 140s loop.

A Bicistronic Plasmid Encoding Brain-Derived Neurotrophic Factor and Urokinase Plasminogen Activator Stimulates Peripheral Nerve Regeneration After Injury.

Timely nerve restoration is an important factor for the successful regeneration of tissues and organs. It is known that axon regeneration following nerve injury is a multifactorial process that depends on the local expression of neurotrophins, including brain-derived neurotrophic factor (BDNF). Along with the survival of neurons, the active reorganization of the extracellular matrix is an important step for the growth of axons to their targets. Urokinase serine protease is part of the plasminogen activator system, which provides the vectoriality of the process of fibrinolysis and matrix reorganization, facilitating the growth of nerves to their targets. Based on this and in view of the results of our previous studies, we suggest that a combined bicistronic plasmid encoding the complementary proteins BDNF and urokinase may be beneficial in nerve regeneration. The ability of this bicistronic plasmid to stimulate nerve restoration was confirmed by in vitro stimulation of Neuro2a neurite growth and in vivo nerve conductivity and histology studies. To our knowledge, this is the first article that demonstrates the effectiveness of a bicistronic plasmid containing the human genes BDNF and urokinase plasminogen activator in the regeneration of the injured peripheral nerve. The results obtained demonstrate that plasmid vectors encoding several complementary-active therapeutic proteins may serve as a basis for developing prospective treatments for a wide range of multicomponent neural system disorders, such as nerve trauma. SIGNIFICANCE STATEMENT: This study is the first to show the effectiveness of using a bicistronic plasmid encoding complementary-active human protein brain-derived neurotrophic factor and urokinase plasminogen activator in the regeneration of the crushed peripheral nerve in a murine model.

Prevention of Anastomotic Leak Via Local Application of Tranexamic Acid to Target Bacterial-mediated Plasminogen Activation: A Practical Solution to a Complex Problem.

To investigate the role of bacterial- mediated plasminogen (PLG) activation in the pathogenesis of anastomotic leak (AL) and its mitigation by tranexamic acid (TXA). AL is the most feared complication of colorectal resections. The pathobiology of AL in the setting of a technically optimal procedure involves excessive submucosal collagen degradation by resident microbes. We hypothesized that activation of the host PLG system by pathogens is a central and targetable pathway in AL. We employed kinetic analysis of binding and activation of human PLG by microbes known to cause AL, and collagen degradation assays to test the impact of PLG on bacterial collagenolysis. Further, we measured the ability of the antifibrinolytic drug TXA to inhibit this process. Finally, using mouse models of pathogen-induced AL, we locally applied TXA via enema and measured its ability to prevent a clinically relevant AL. PLG is deposited rapidly and specifically at the site of colorectal anastomoses. TXA inhibited PLG activation and downstream collagenolysis by pathogens known to have a causal role in AL. TXA enema reduced collagenolytic bacteria counts and PLG deposition at anastomotic sites. Postoperative PLG inhibition with TXA enema prevented clinically and pathologically apparent pathogen-mediated AL in mice. Bacterial activation of host PLG is central to collagenolysis and pathogen-mediated AL. TXA inhibits this process both in vitro and in vivo. TXA enema represents a promising method to prevent AL in high-risk sites such as the colorectal anastomoses.

Designing a multifunctional staphylokinase variant (SAK-2RGD-TTI) with appropriate thrombolytic activity in vitro.

Thrombin, platelets, and plasmin are three key factors involved in hemostasis and thrombolysis. Thrombolytic therapy with clinically approved drugs is often followed by recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, new constructs were designed for the expression of recombinant staphylokinase (rSAK) and also a fusion protein composed of staphylokinase, 20 amino acids containing 2 RGD followed by tsetse thrombin Inhibitor (SAK-2RGD-TTI) in Pichia pastoris. Modeling the tertiary structure of SAK-2RGD-TTI showed that the linker containing RGD and TTI did not interfere with proper folding of SAK. In laboratory testing, the purified SAK-2RGD-TTI (420μg/mL) dissolved an average of 45% of the blood clot. The activity of the SAK-2RGD-TTI was also confirmed in various tests including human plasminogen activation assay, fibrin clot lysis assay, well diffusion method, activated partial thromboplastin time and platelet rich clot lysis assay. Our findings suggest that SAK-2RGD-TTI has improved therapeutic properties preventing reocclussion. It further confirms that it is practicable to assemble and produce a hybrid multifunctional protein that targets hemostatic process at various stages.

Accurately cleavable goat β-lactoglobulin signal peptide efficiently guided translation of a recombinant human plasminogen activator in transgenic rabbit mammary gland.

Poor expression is the key factor hampering the large-scale application of transgenic animal mammary gland bioreactors. A very different approach would be to evaluate the secretion of recombinant proteins into milk in response to a cleavable signal peptide of highly secreted lactoproteins.We previously reported rabbits harboring mammary gland-specific expression vector containing a fusion cDNA (goat β-lactoglobulin (BLG) signal peptide and recombinant human plasminogen activator (rhPA) coding sequences) expressed rhPA in the milk, but we did not realize the signal peptide contributed to the high rhPA concentration and did not mention it at that time. And the molecular structure and biological characteristics still remain unknown. So, rhPA in the milk was purified and characterized in the present study.rhPA was purified from the milk, and the purity of the recovered product was 98% with no loss of biological activity. Analysis of the N-terminal sequence, C-terminal sequence, and the molecular mass of purified rhPA revealed that they matched the theoretical design requirements. The active systemic anaphylaxis (ASA) reactions of the purified rhPA were negative. Taken together, these results indicated that the goat BLG signal peptide can efficiently mediate rhPA secretion into milk and was accurately cleaved off from rhPA by endogenous rabbit signal peptidase.We have reinforced the importance of a rhPA coding region fused to a cleavable heterologous signal peptide from highly secreted goat BLG to improve recombinant protein expression. It is anticipated that these findings will be widely applied to high-yield production of medically important recombinant proteins.

Transient expression of bioactive recombinant human plasminogen activator in tobacco leaf

To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production. Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by in vitro sequence synthesis and subcloning. The two vectors were inoculated on either Nicotiana benthamiana or N. excelsiana leaves via agroinfiltration. The expression of recombinant rhPA in Nicotiana leaves was examined using Western blotting and ELISA, and the in vitro fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA). Five to nine days after infiltration with an Agrobacterium inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both Nicotiana plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated N. benthamiana leaves was significantly higher than that in N. excelsiana leaves (P &lt; 0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both Nicotiana leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles. Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.

6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer.

Metastasis is the cause of death in the majority (∼90%) of malignant cancers. The oral potassium-sparing diuretic amiloride and its 5-substituted derivative 5 -N, N-(hexamethylene)amiloride (HMA) reportedly show robust antitumor/metastasis effects in multiple in vitro and animal models. These effects are likely due, at least in part, to inhibition of the urokinase plasminogen activator (uPA), a key protease determinant of cell invasiveness and metastasis. This study reports the discovery of 6-substituted HMA analogs that show nanomolar potency against uPA, high selectivity over related trypsin-like serine proteases, and minimal inhibitory effects against epithelial sodium channels (ENaC), the diuretic and antikaliuretic target of amiloride. Reductions in lung metastases were demonstrated for two analogs in a late-stage experimental mouse metastasis model, and one analog completely inhibited formation of liver metastases in an orthotopic xenograft mouse model of pancreatic cancer. The results support further evaluation of 6-substituted HMA derivatives as uPA-targeting anticancer drugs.

A novel recombinant human plasminogen activator: Efficient expression and hereditary stability in transgenic goats and in vitro thrombolytic bioactivity in the milk of transgenic goats.

BackgroundThromboses is a rapidly growing medical problem worldwide. Low-cost, high-scale production of thrombotic drugs is needed to meet the demand. The production of biomolecules in transgenic animals might help address this issue. To our knowledge, the expression of recombinant human plasminogen activator (rhPA) in goat mammary glands has never been reported before.MethodsWe constructed a mammary gland–specific expression vector, BLC14/rhPA, which encodes only the essential K2 fibrin-binding and P domains of wild-type tPA (deletion mutant of tPA lacking the F, E, and K1 domains), along with the goat β-lactoglobulin gene signal peptide-coding sequence. The mammary gland–specific expression vector BLC14/rhPA was transfected into goat fetal fibroblast cells by electroporation. After selection for 3 weeks by G418, stably transfected cell colonies were obtained. PCR analysis results indicated that 24 of the resistant clones were transgenic cell lines; of these, 8 lines were selected as the donor cells. The positive cells were starved for 72 h with DMEM/F12 medium containing 0.5% FBS and were then used as do. Finally, 256 reconstructed oocytes were transferred into 26 recipients, and 7 of them became pregnant (pregnancy rate, 26.9%). Two kids were obtained (BP21 and BP22). PCR analysis confirmed that both were transgenic goats. To analyze the heredity of the rhPA expressed in BP21 F0 and F1 transgenic goats, the F0 transgenic goat BP21 was mated with a normal male goat to generate an F1 transgenic goat. Enucleated metaphase II (MII) oocytes and positive donor cells were used to reconstruct embryos, which were transplanted into the oviducts of the recipients.ResultsWestern blot results showed a specific 39 kDa band. The rhPA expression level in transgenic goat whey was about 78.32 μg/mL by ELISA. Results of ELISA and the in vitro thrombolysis test (FAPA) showed that specific activity of the rhPA in the milk of F0 and F1 transgenic goats was 13.3 times higher than that of the reteplase reference material.ConclusionThus, we demonstrated that BLC14/rhPA was reasonably effective for expression in the mammary glands of transgenic goats, and was stably inherited by the offspring. This study provides the basis for the large-scale production of biological pharmaceuticals in transgenic animals. The expression of biopharmaceuticals by transgenic animals can be used for pharmacological research and bioactive analysis, and transgenic goats were demonstrated to be promising animals for the large-scale production of thrombolytic biopharmaceuticals.

Efficient increase of the novel recombinant human plasminogen activator expression level and stability through the use of homozygote transgenic rabbits.

Expression efficacy of recombinant protein in current expression systems is generally low. Therefore, the expression levels of recombinant proteins in the breast milk of transgenic animals are typically low. In view of this, the present study aimed to construct homozygous transgenic rabbits with a high expression level of recombinant human plasminogen activator (rhPA) during the entire lactation period. Homozygous transgenic rabbits were obtained using an effective rhPA mammary‑specific expression vector PCL25/rhPA. The expression level and thrombolytic ability of rhPA in the milk of both homozygous and hemizygous rabbits were detected by enzyme‑linked immunosorbent and fibrin agarose plate assays. It was observed that the expression of rhPA was constant during the entire lactation period in homozygous rabbits, while the expression of rhPA declined slowly in hemizygote rhPA transgenic rabbits during the lactation period. In addition, the expression of rhPA in homozygous transgenic rabbit was ~950µg/ml, which was markedly higher in comparison with that in hemizygote rabbits. Furthermore, increased gene copy number was observed to increase the expression level of rhPA at the same integration vector.

Famotidine as Urokinase Inhibitor in Anaphylactic Shock

A QSAR study was performed on a series of naphthalene cyclohexane amines derivate with experimentally determine constant of inhibition (Ki ) in order to indentify a potent human urokinase inhibitor. A QSAR model was build using artificial neural networks (ANN). Model was used for screening of compounds with potent urokinase inhibitory properties and to compute Ki of resulted screening compounds. Compound with best Ki was chosen for discussion. An induced fit docking (protein - ligand) and a protein –protein docking were used in results discussion. A isoquinoline –diol base derivate (Famotidione) resulted to have urokinase inhibitory properties.

D2A-Ala peptide derived from the urokinase receptor exerts anti-tumoural effects in vitro and in vivo

D2A-Ala is a synthetic peptide that has been created by introducing mutations in the original D2A sequence, 130IQEGEEGRPKDDR142 of human urokinase receptor (uPAR). In vitro, D2A-Ala peptide displays strong anti-tumoural properties inhibiting EGF-induced chemotaxis, invasion and proliferation of a human fibrosarcoma cell line, HT 1080, and a human colorectal adenocarcinoma cell line, HT 29. D2A-Ala exerts its effects by preventing EGF receptor (EGFR) phosphorylation.To test D2A-Ala in vivo, this peptide was PEGylated generating polyethyleneglycol (PEG)-D2A-Ala peptide. PEGylation did not alter the inhibitory properties of D2A-Ala. Human tumour xenografts in the immunodeficient nude mice using HT 1080 and HT 29 cell lines showed that PEG-D2A-Ala significantly prevents tumour growth decreasing size, weight and density of tumours. The most efficient doses of the peptide were 5 and 10 mg/kg, thereby relevant for possible development of the peptide into a drug against cancer in particular tumours expressing EGFR.

Cavity Versus Ligand Shape Descriptors: Application to Urokinase Binding Pockets.

We analyzed 78 binding pockets of the human urokinase plasminogen activator (uPA) catalytic domain extracted from a data set of crystallized uPA–ligand complexes. These binding pockets were computed with an original geometric method that does NOT involve any arbitrary parameter, such as cutoff distances, angles, and so on. We measured the deviation from convexity of each pocket shape with the pocket convexity index (PCI). We defined a new pocket descriptor called distributional sphericity coefficient (DISC), which indicates to which extent the protein atoms of a given pocket lie on the surface of a sphere. The DISC values were computed with the freeware PCI. The pocket descriptors and their high correspondences with ligand descriptors are crucial for polypharmacology prediction. We found that the protein heavy atoms lining the urokinases binding pockets are either located on the surface of their convex hull or lie close to this surface. We also found that the radii of the urokinases binding pockets and the radii of their ligands are highly correlated (r = 0.9).

Molecular Effects of Stromal-Selective Targeting by uPAR-Retargeted Oncolytic Virus in Breast Cancer.

The tumor microenvironment (TME) is a relevant target for novel biological therapies. MV-m-uPA and MV-h-uPA are fully retargeted, species-specific, oncolytic measles viruses (MV) directed against murine or human urokinase receptor (PLAUR/uPAR), expressed in tumor and stromal cells. The effects of stromal-selective targeting by uPAR-retargeted MVs were investigated. In vitro infection, virus-induced GFP expression, and cytotoxicity by MV-h-uPA and MV-m-uPA were demonstrated in human and murine cancer cells and cancer-associated fibroblasts in a species-specific manner. In a murine fibroblast/human breast cancer 3D coculture model, selective fibroblast targeting by MV-m-uPA inhibited breast cancer cell growth. Systemic administration of murine-specific MV-m-uPA in mice bearing human MDA-MB-231 xenografts was associated with a significant delay in tumor progression and improved survival compared with controls. Experiments comparing tumor (MV-h-uPA) versus stromal (MV-m-uPA) versus combined virus targeting showed that tumor and stromal targeting was associated with improved tumor control over the other groups. Correlative studies confirmed in vivo viral targeting of tumor stroma by MV-m-uPA, increased apoptosis, and virus-induced differential regulation of murine stromal genes associated with inflammatory, angiogenesis, and survival pathways, as well as indirect regulation of human cancer pathways, indicating viral-induced modulation of tumor-stroma interactions. These data demonstrate the feasibility of stromal-selective targeting by an oncolytic MV, virus-induced modulation of tumor-stroma pathways, and subsequent tumor growth delay. These findings further validate the critical role of stromal uPAR in cancer progression and the potential of oncolytic viruses as antistromal agents.Implications: The current report demonstrates for the first time the biological, in vitro, and in vivo antitumor and molecular effects of stromal selective targeting by an oncolytic virus. Mol Cancer Res; 15(10); 1410-20. ©2017 AACR.

Recombinant human TNK tissue-type plasminogen activator (rhTNK-tPA) versus alteplase (rt-PA) as fibrinolytic therapy for acute ST-segment elevation myocardial infarction (China TNK STEMI): protocol for a randomised, controlled, non-inferiority trial

AimTo evaluate the efficacy and safety of recombinant human TNK tissue-type plasminogen activator (rhTNK-tPA) in lowering major adverse cardiovascular and cerebrovascular events (MACCEs) in Chinese acute ST-segment elevation myocardial infarction...

Abstract 5093: In vitro, in vivo and molecular effects of stromal selective targeting by uPAR retargeted oncoytic measles virus on breast cancer progression

Abstract The tumor microenvironment is a relevant target for novel biological therapies. While it has been demonstrated that tumor-stromal cell interactions are important in the sensitivity of the cancer cells to oncolytic virues, few studies have investigated the direct effects of an oncolytic virus on the stroma, and its implications on the virus antitumor efficacy. MV-m-uPA and MV-h-uPA are fully retargeted, species specific, oncolytic measles viruses directed against murine or human urokinase receptor (uPAR). The effects of stromal selective targeting by uPAR retargeted MVs were investigated. In vitro infection, gene expression and cytotoxicity by MV-h-uPA and MV-m-uPA were demonstrated in human and murine cancer cells and cancer associated fibroblasts in a species specific manner. In murine fibroblast (3T3)/human breast cancer (T47D) 3-D co-cultures, selective fibroblast targeting by MV-m-uPA inhibited breast cancer cell growth. These effects were validated in vivo, in a human breast cancer xenograft (MDA-MB-231), where intravenous administration of the murine specific MV-m-uPA led to significant tumor growth delay and improved survival compared to controls. Experiments comparing the effects of tumor (MV-h-uPA) vs. stromal (MV-m-uPA) vs. combined treatment showed that tumor and stromal targeting was associated with improved tumor control. Correlative studies demonstrated in vivo tumor targeting, increased apoptosis, and MV-m-uPA induced differential regulation of both stromal (murine) genes and cancer (human) genes associated with inflammation, angiogenesis and survival, among others, indicating viral induced modulation of tumor-stromal interactions. Our results demonstrate for the first time the feasibility of stromal selective targeting by an oncolytic MV, viral induced modulation of the tumor microenvironment, and subsequent tumor growth delay. These findings further validate the critical role of stromal uPAR in cancer progression and the potential of oncolytic viruses as anti-stromal agents. Citation Format: Yuqi Jing, Valery Chavez, Nicolas Acquavella, Doraya El-Ashry, Yuguang Ban, Xi Chen, Jaime Merchan. In vitro, in vivo and molecular effects of stromal selective targeting by uPAR retargeted oncoytic measles virus on breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2017-5093

Clinical efficacy of sequential therapy for butylphthalide combined with alteplase in the treatment of acute cerebral infarction

Objective To observe the effect of butylphthalide sequential therapy combined with injection of alteplase(injection with recombinant human collagenous tissue plasminogen activator) in the treatment of acute cerebral infarction (ACI). Methods 61 patients with ACI and within 4.5 hours of onset were selected, and they were divided into treatment group(butylphthalide combined with alteplase group, 31 cases) and the control group (alteplase only group, 30 cases) by random number table.The two groups were given glucose-lowering, statins for maintaining the blood pressure, as well as aspirin enteric tablets or clopidogrel for those without bleeding on the 24 hour review head CT in the acute phase.The treatment group was given alteplase for injection with sequential butylphthalide therapy (butylphthalide injection for 2 weeks followed by an oral administration of butylphthalide soft capsules for 3 months), and the control group was given only alteplase for injection.The National Institute of Health stroke scale (NIHSS) and Barthel index (BI) were used for evaluating the final clinical efficacy. Results The NIHSS scores of the treatment group compared with the control group at 24 hour, 14 days and 3.5 months after the treatment had statistically significant differences(t=-0.102, -1.964, -2.444, P=0.037, 0.018 and 0.012). The NIHSS scores of the treatment group had decreased 2%, 27% and 41% compared with the control group at 24 hour, 14 days and 3.5 months after the treatment, respectively.The BI score of the treatment group and control group at 3.5 months after treatment had statistically significant difference(t=-1.956, P=0.029). Conclusion The sequential butylphthalide therapy combined with injection of alteplase has good synergistic effect for the patients with onset of ACI less than 4.5 hours, and it is a safe and effective treatment method for ACI within 4.5 h and it is worthy of clinical promotion. Key words: Cerebral infarction, acute; Butylphthalide; Alteplase