Abstract WP285: tPA Variant tPA-A296-299 Prevents Impairment of Cerebral Autoregulation and Hippocampal Neuronal Necrosis After Stroke Through Inhibition of ET-1 Upregulation

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
William Armstead ◽  
Hugh Hekierski ◽  
Serge Yarovoi ◽  
Abd Al-Roof Higazi ◽  
Douglas Cines
Keyword(s):  
Cerebral Autoregulation ◽  
Neuronal Cell ◽  
Tissue Type ◽  
Therapeutic Window ◽  
Dependent Manner ◽  
Wild Type ◽  
Neuronal Necrosis ◽  
Cranial Window ◽  
Type Plasminogen Activator

Introduction: The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, its brief therapeutic window and post-treatment complications markedly constrain its use. The limited efficacy of tPA may be explained in part by our finding that it potentiates impairment of cerebral autoregulation after stroke in pigs. Upregulation of JNK MAPK after thrombotic stroke contributes to tPA mediated impairment of autoregulation. However, the role of ET-1, and its relationship to JNK, in impairment of autoregulation is unknown. Impairment of autoregulation is linked to Glasgow Coma Scale after brain injury, but unknown if autoregulation correlates with hippocampal histopathology after stroke. Hypothesis: Wild type (wt) tPA can bind to either the lipoprotein-related receptor (LRP), which mediates vasodilation, or NMDA-Rs. We propose that wt tPA given after stroke primarily binds to NMDA-Rs, upregulates ET-1 in a JNK dependent manner, which impairs autoregulation, causing histopathology. tPA-A 296-299 , a variant that is fibrinolytic but cannot bind to NMDA-Rs, preferentially binds LRP, which blocks ET-1 and JNK upregulation and limits impairment of autoregulation and histopathology after stroke. Methods: Anesthetized pigs equipped with a closed cranial window were used. Stroke was induced by photothrombosis, CBF determined by radiolabeled microspheres, CSF ET-1 and JNK determined by ELISA and histopathology determined with H + E stain. Results: CBF was unchanged during hypotension (mean arterial pressure decreased by 45%) in sham animals. CBF was reduced in brain tissue surrounding the infarct and was reduced further during hypotension, indicating impairment of autoregulation. CA1 and CA3 neuronal cell necrosis and autoregulation were further impaired by wt tPA which was prevented by BQ 123 and tPA-A 296-299 . Cerebral ET-1 was increased by stroke, potentiated by wt tPA, but returned to sham baseline value by tPA-A 296-299 and the JNK antagonist SP600125. Conclusions: JNK contributes to ET-1 release after stroke. tPA-A 296-299 prevents impairment of cerebral autoregulation and histopathology after stroke through inhibition of ET-1 upregulation.

Abstract 172: tPA Variant tPA-A296-299 Prevents Impairment of Cerebral Autoregulation After Stroke Through LRP Dependent Increase in cAMP and p38 MAPK

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
William Armstead ◽  
John Riley ◽  
Serge Yarovoi ◽  
Abd Al-Roof Higazi ◽  
Douglas Cines
Keyword(s):  
Cerebral Autoregulation ◽  
Tissue Type ◽  
Therapeutic Window ◽  
Wild Type ◽  
Tissue Type Plasminogen Activator ◽  
P38 Inhibitor ◽  
Cranial Window ◽  
Limited Efficacy ◽  
Type Plasminogen Activator ◽  
Sb 203580

Introduction: The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, its brief therapeutic window and post-treatment complications markedly constrain its use. The limited efficacy of tPA may be explained in part by our finding that it potentiates impairment of cerebral autoregulation after stroke in pigs. Upregulation of JNK MAPK after thrombotic stroke contributes to tPA mediated impairment of autoregulation whereas p38 is protective. Vasodilation is mediated by elevation of cAMP. However, cAMP falls after stroke due to over activation of NMDA receptors (NMDA-Rs) by toxic levels of glutamate, which is exacerbated by tPA. Hypothesis: Wild type (wt) tPA can bind to either the lipoprotein-related receptor (LRP), which mediates vasodilation, or NMDA-Rs. We propose that wt tPA given after stroke primarily binds to NMDA-Rs, reduces cAMP, which impairs ability of cerebral vessels to vasodilate. tPA-A296-299, a variant that is fibrinolytic but cannot bind to NMDA-Rs, preferentially binds LRP, which increases cAMP and p38 and limits impairment of autoregulation after stroke. Methods: Anesthetized pigs equipped with a closed cranial window were used. Stroke was induced by photothrombosis, CBF determined by radiolabeled microspheres, and CSF cAMP and p38 determined by ELISA. Results: CBF was unchanged during hypotension (mean arterial pressure decreased by 45%) in sham animals. CBF was reduced in brain tissue surrounding the infarct and was reduced further during hypotension, indicating impairment of autoregulation. Autoregulation was further impaired by wt tPA which was prevented by MK801 and tPA-A296-299. Protection by tPA-A296-299 was blocked by anti LRP Ab, the LRP antagonist RAP, the p38 inhibitor SB 203580, but not by IgG. Stroke reduced CSF cAMP, which was reduced further by wt tPA, but augmented by tPA-A296-299. CSF p38 was unchanged by wt tPA, robustly increased by tPA-A296-299, and decreased by co-administered anti LRP Ab and RAP but not by IgG. Conclusions: tPA-A296-299 prevents impairment of cerebral autoregulation after stroke through LRP dependent increase in cAMP and p38.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

Role of plasminogen activators in peritoneal adhesion formation

2002 ◽  
Vol 30 (2) ◽  
pp. 126-131 ◽  
Author(s):  
H. Sulaiman ◽  
L. Dawson ◽  
G. J. Laurent ◽  
G. J. Bellingan ◽  
S. E. Herrick
Keyword(s):  
Plasminogen Activator ◽  
Adhesion Formation ◽  
Tissue Type ◽  
Surgical Trauma ◽  
Wild Type ◽  
Post Injury ◽  
Peritoneal Adhesion ◽  
Type Plasminogen Activator ◽  
Peritoneal Adhesion Formation

Intra-abdominal adhesion formation is a major complication of serosal repair following surgery, ischaemia or infection, leading to conditions such as intestinal obstruction and infertility. It has been proposed that the persistence of fibrin, due to impaired plasminogen activator activity, results in the formation of adhesions between damaged serosal surfaces. This study aimed to assess the role of fibrinolysis in adhesion formation using mice deficient in either of the plasminogen activator proteases, tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). We hypothesize that, following serosal injury, mice with decreased peritoneal fibrinolytic activity will be more susceptible to adhesion formation. Adhesion formation was induced in tPA- and uPA-deficient and wild-type mice following either surgical trauma to the serosa with haemorrhage and acute or chronic intraperitoneal inflammation. Adhesion formation was assessed from 1 to 4 weeks post-injury. Mice deficient in tPA were more susceptible to adhesion formation following both a surgical insult and a chronic inflammatory episode compared with uPA-deficient and wild-type mice. In addition, the time of maximal adhesion formation varied depending on the nature of the initial insult. It is proposed that the persistence of fibrin due to decreased tPA activity following surgery or chronic inflammation plays a major role in peritoneal adhesion formation.

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA

1990 ◽  
Vol 64 (01) ◽  
pp. 061-068 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoet ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Active Site ◽  
Peptide Bond ◽  
Tissue Type ◽  
Wild Type ◽  
Single Chain ◽  
Plasmin Activity ◽  
Soluble Fibrin ◽  
Type Plasminogen Activator ◽  
Presence And Absence ◽  
Chain Form

SummaryThe activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant plasminogen with the active site Ser740 mutagenaed to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA -Glul275, a recombinant single chain t-PA in which the Arg of the plasmin sensitiv e Arg275- Ile276 peptide bond was substituted with Glu. Conversion of 125I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v0 in pM s−1) per 1 μM enzyme. In the absence of fibrin stimulation, the vs for the activation of nPlg and rPlg-Ala740 with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s−1) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s−1). In the presence of 1 μM soluble fibrin monomer (desAAfibrin), the v0 for nPlg and rPlg-Ala740 by single chain rt-PA was also comparable (24 and, 33 pM s-1 respectively), whereas with 1 pM CNBr-digested fibrinogen, the vs for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala740 (135 and 7.5 pM s−1 respectively). In contrast, the vs for nPlg and rPlg-Ala740 by single chain rt-PA- G1u275, two-chain rt-PA-G1u275 or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.These findings confirm and establish: 1) that single chain t-PA is an active enzyme both in the presence and absence of fibrin stimulator; 2) that, in a system devoid of plasmin activity (rPlg- Ala740), the two-chain form of t-PA is about L5 times more active than the single chain form in the absence of fibrin but equipotent in the presence of desAAfibrin; and 3) that the mechanism of stimulation of plasminogen activation with single chain t-PA by CNBr-digested fibrinogen is different from that by soluble fibrin.

Increased tissue-type plasminogen activator activity in orthotopic but not heterotopic liver transplantation: The role of the anhepatic period

Hepatology ◽  
1992 ◽  
Vol 16 (2) ◽  
pp. 404-408 ◽  
Author(s):  
C. Minke Bakker ◽  
Herold J. Metselaar ◽  
Theo N. Groenland ◽  
Maria J. Gomes ◽  
Eduard A. R. Knot ◽  
...  
Keyword(s):  
Liver Transplantation ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Plasminogen Activator Activity ◽  
Type Plasminogen Activator ◽  
Anhepatic Period

Abstract 2579: tPA Contributes To Aggravation of Endothelin and Thromboxane Induced Cerebrovasoconstriction After Hypoxia/Ischemia Through Upregulation of ERK MAPK

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
William Armstead ◽  
Heather Kaczynski ◽  
John Riley ◽  
Douglas Cines ◽  
Abd Al-Roof Higazi
Keyword(s):  
Mitogen Activated Protein Kinase ◽  
Tissue Type ◽  
Cranial Window ◽  
Pial Artery ◽  
Erk Mapk ◽  
Cerebral Vasoconstriction ◽  
Type Plasminogen Activator ◽  
Hypoxia Ischemia ◽  
Mitogen Activated Protein ◽  
Global Increase

Introduction: The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, endogenous tPA is upregulated and potentiates impairment of pial artery dilation in response to hypotension after hypoxia/ischemia (H/I) in pigs. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is also upregulated after H/I, with ERK contributing to vasodilator impairment. Impairment of stimulus induced vasodilation may result from tonic withdrawal and/or impairment of a vasodilator influence or upregulation of a vasoconstrictor. This study examined the effect of H/I on the vascular response to two important spasmogens released during CNS ischemic disorders, endothelin-1 (ET-1) and thromboxane, and the influence of tPA and ERK MAPK in such responses. Methods: Cerebral ischemia (20 min) was induced via global increase in intracranial pressure via saline infusion into a hollow bolt placed in the cranium (dura intact) while hypoxia (10 min, pO2 35 mm Hg) was produced by decreasing the inspired O2 via inhalation of N2. Vascular responses to topical ET-1 (10-10, 10-8 M) and U 46619 (1, 10 ng/ml) were obtained at 1h post insult. Phosphorylated and total ERK MAPK were measured by ELISA in the CSF of piglets equipped with a closed cranial window. Data (n=5) were analyzed by ANOVA, with significance at p less than 0.05. Results: H/I aggravated pial artery vasconstriction induced by ET-1 and the thromboxane mimic U 46619, which was blocked by EEIIMD, an inhibitor of PAI-1’s vascular activity and signaling of tPA, but not its fibrinolytic action, but unchanged by its inactive analogue EEIIMR. CSF ERK MAPK was increased by H/I and potentiated by tPA. The ERK MAPK antagonist U 0126 blocked H/I induced aggravation of ET-1 and U 46619 vasoconstriction. Discussion: These data indicate that H/I aggravates ET-1 and thromboxane mediated cerebral vasoconstriction through upregulation of tPA and ERK MAPK. These data suggest that thrombolytic therapy for treatment of CNS ischemic disorders can dysregulate cerebrohemodynamics by augmenting vasoconstriction induced by spasmogens co-released during CNS pathology.

Urokinase-type plasminogen activator receptor is associated with the development of adipose tissue

2010 ◽  
Vol 104 (12) ◽  
pp. 1124-1132 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Eri Kawashita ◽  
Kiyotaka Okada ◽  
Hidetaka Suga ◽  
Shigeru Ueshima ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Plasminogen Activator ◽  
Adipocyte Differentiation ◽  
Cellular Adhesion ◽  
Wild Type ◽  
Tissue Mass ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Plasminogen Activator Receptor

SummaryUrokinase-type plasminogen activator receptor (uPAR) plays a role in cellular responses which include cellular adhesion, differentiation, proliferation and migration. The aim of this study was to clarify the role of uPAR on the development of adipose tissue. To clarify the role of uPAR on adipogenesis, we examined the effect of uPAR overexpression and uPAR deficiency on the adipocyte differentiation. Adipocyte differentiation was induced by incubation of 3T3-L1 cells with differentiation media containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthin. uPAR overexpression by transfection of uPAR expression vector induced adipocyte differentiation. In addition, we examined the difference in adipocyte differentiation of mesenchymal stem cells from wild-type mice and uPAR knockout (uPAR-/-) mice. The uPAR deficiency attenuated differentiation media-induced adipocyte differentiation. Moreover, we found that the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway attenuated uPAR overexpression-induced adipocyte differentiation, and uPAR overexpression induced the activation of Akt. We also found that an increase of the adipose tissue mass in uPAR-/- mice was less than that observed in wild-type mice. The present results suggest that uPAR plays a pivotal role in the development of adipose tissue through PI3K/Akt pathway.

scholarly journals CLL-Derived Exosomes Function As Trojan Horses That Induce SMAD6-Dependent Apoptosis of Normal B-Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1734-1734
Author(s):  
Orit Uziel ◽  
Zinab Sarsur- Amer ◽  
Einat Beery ◽  
Pia Raanani ◽  
Uri Rozovski
Keyword(s):  
B Cells ◽  
Time Dependent ◽  
Dependent Manner ◽  
Environmental Resources ◽  
Type B ◽  
Wild Type ◽  
Cell Competition ◽  
Western Immunoblotting ◽  
Competition Theory

Studies from recent years unraveled the role of monocytes and T-cells in the pathogenesis of chronic lymphocytic leukemia (CLL). The role of other immune cells in the pathobiology of CLL is less known. Specifically, whether B-cells, the normal counterpart of CLL cells play a role in CLL is unknown. Nevertheless, since both CLL cells and wild type B-cells reside in lymphatic organs and travel in blood, they either share or compete over common environmental resources. According to the cell competition theory, a sensing mechanism measures the relative fitness of a cell and ensures the elimination of cells deemed to be less fit then their neighbors. Since constitutive activation of intracellular pathways protect CLL cells from apoptosis, the cell competition theory predicts that compared with normal B-cells these cells are sensed as "super fit" and B-cells, the less fit counterparts, are eliminated. Yet, what delivers this massage across a population of cells is unknown. Exosomes are nanosized particles that are secreted by various types of cells. Exosomes carry a cargo of proteins and different types of RNA. They travel in body fluids and are taken up by cells in their vicinity. Since cancer cells including CLL cells secrete exosomes, we have formulated our hypothesis, namely, that exosomes derived from CLL cells are the vehicles that carry a death massage to wild type B-cells. To test this hypothesis, we isolated CLL cells from 3 previously untreated patients with CLL. We then grew these cells in exosome free media for 72 hours and harvested the exosomes by ultracentrifugation. We used NanoSight tracking analysis, Western immunoblotting for CD63, a common exosomal marker, and electron microscopy imaging studies to ensure that our pellet include the typical 100nm exosomal particles. Subsequently, we subjected normal B-cells derived from healthy volunteers to CLL derived exosomes stained by FM-143 dye. Using flow cytometry we found that exosomes are taken up by normal B-cells in a dose- and time- dependent manner. Double staining of the recipient B-cells to Annexin/PI revealed that exosomes induce apoptosis of these cells in a dose- and time- dependent manner. We then used RNA-seq to trace the changes in the molecular makeup of B-cells after exosomal uptake?? they took up exosomes. We found 24 transcripts that were differentially expressed (11 that were upregulated and 13 that were downregulated). We then verified the array results by quantitative real-time PCR for four of these genes. Among the top transcripts that were upregulated in exosome-positive B-cells is SMAD6. Because the upregulation of the SMAD family members including SMAD6 is associated with the induction of apoptosis in various malignant and non-malignant cells we wondered whether the upregulation of SMAD6 also induces apoptosis in normal B-cells. To test this, we transfected normal B-cells with SMAD6 containing vector and verified by RT-PCR that level of SMAD6 transcript were upregulated and by Western immunoblotting that levels of SMAD6 protein are upregulated as well. As expected, the rate of apoptosis was higher, and the rates of viable cells and proliferating cells were significantly lower in SMAD6-transfected B-cells. Taken together, we show here that CLL cells secrete exosomes that function as "Trojan horses". Once they are taken up by normal B-cells they induce SMAD6-dependent apoptosis. In this way the neoplastic cells may actively eliminate their competitors and take over the common environmental resources. Disclosures No relevant conflicts of interest to declare.

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