The W–x–x–W Motif in the TSR1 of ADAMTS13 Is Important for Its Secretion

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4389-4389
Author(s):  
Jing Ling ◽  
Jian Su ◽  
Zhenni Ma ◽  
Changgeng Ruan
Keyword(s):  
Shear Stress ◽  
Binding Affinity ◽  
Culture Medium ◽  
Stress Condition ◽  
Conflicts Of Interest ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Shear ◽  
High Shear Stress ◽  
Wild Type ◽  
Western Blots

Abstract Abstract 4389 Introduction: The W-x-x-W motif is commonly found in the thrombospondin type 1 repeat (TSR) of various extra cellular proteins called TSR super family proteins, including thrombospondins, ADAMTSs, F-spondin and properdin. The W–x–x–W motif is known to bind with heparin, heparan sulfate proteoglycans, collagen and transforming growth factor-β, suggesting functional significance in cell–cell interaction and/or cellular signaling. However, the function of W-x-x-W in ADAMTS13 is unclear. In this study, we investigated the role of the W-x-x-W motif of ADAMTS13. Materials and Methods: We generated a W-x-x-W mutant (W387A) construct of ADAMTS13, and expressed the mutant and the wild-type constructs in HELA cells. Percentage of the protein secretion was defined as the concentration in the culture medium divided by the concentrations in the culture medium and cell lysates, multiplied by 100%. The binding affinity of the mutant or wild-type ADAMTS13 was investigated by enzyme-linked immunosorbent assay. Measurement of ADAMTS13 proteolytic activity toward von Willebrand factor (VWF) multimers was based on the generation of a dimeric 176-kDa fragment resulting from cleavage of VWF at the Y1605-M1606 bond, under denaturing condition and high shear stress condition, analyzed by Western blots. Results: SDS-PAGE gel analysis showed that the W387A mutant was secreted less efficiently relative to the wild-type construct. As for the binding affinity for the VWF multimer, there was no difference between the wild-type and mutant ADAMTS13. The W387A mutant was less active under denaturing condition; the same result was reproduced when FRETS-VWF73 was used as the substrate. However, under high shear stress condition, the mutant was as efficient as the wild-type ADAMTS13. Conclusions: The W–x–x–W motif is necessary for efficient secretion of ADATMS13. Further studies are needed to determine the contribution of the motif to the VWF cleave activity of ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High shear stress suppresses proliferation and migration but promotes apoptosis of endothelial cells co-cultured with vascular smooth muscle cells via down-regulating MAPK pathway

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Qiang Ji ◽  
Yu Lin Wang ◽  
Li Min Xia ◽  
Ye Yang ◽  
Chun Sheng Wang ◽  
...  
Keyword(s):  
Shear Stress ◽  
Culture System ◽  
Stress Condition ◽  
Mapk Pathway ◽  
High Shear ◽  
High Shear Stress ◽  
Cellular Processes ◽  
Mapk Pathways ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background Early neointimal hyperplasia of vein graft may be ameliorated via enhancing intravenous surface shear stress. Cellular processes including proliferation, apoptosis and migration of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) may play very important roles in the process of neointimal hyperplasia of vein graft; and mitogen-activated protein kinase (MAPK) pathways including extracellular signal-regulated kinase (ERK1/2) and p38 pathways play vital roles in regulating a large variety of cellular processes. This study evaluated the impacts of shear stress and MAPK pathways on cellular processes of ECs in a co-culture system with VSMCs, and aimed to test the hypothesis that high shear stress suppresses proliferation and migration but promotes apoptosis of ECs co-cultured with VSMCs via down-regulating MAPK pathway. Methods Primary ECs and VSMCs derived from porcine great saphenous vein were collected, respectively. 4–7 generation of cells were used as work cells. ECs and VSMCs were co-cultured and synchronized under high and low shear stress using Parallel-Plate Flow Chamber system. And then, ECs co-cultured with VSMCs were incubated with U0126 (ERK1/2 inhibitor) or PD98059 (p38 inhibitor) under different shear stress. Proliferation, apoptosis and migration of ECs in a co-culture system with VSMCs were detected by 4,5-dimethyl-2-thiazolyl (MTT) assay and bromodeoxyuridine (BrdU) assay, fluorescent-activated cell sorting (FACS) technique, and Transwell assay separately. Each test repeated 3 times. Additionally, protein expressions of ERK1/2 and p38 MAPK were detected by using Western blot, respectively. Results Under higher level of shear stress condition, proliferation and migration of ECs co-cultured with VSMCs were suppressed, while cell apoptosis was promoted. And blocking ERK1/2 pathway by U0126 or blocking p38 pathway by PD98059, proliferation and migration of ECs co-cultured with VSMCs were further suppressed, while cell apoptosis was further promoted. Additionally, protein expressions of phosphorylation of ERK1/2 and p38MAPK were decreased under higher level of shear stress condition, and were further reduced by blocking ERK1/2 or p38 pathway under shear stress condition. Conclusions High shear stress may suppress proliferation and apoptosis of ECs in a co-culture system with VSMCs but promote cell migration via down-regulating ERK1/2 and p38 MAPK pathways.

scholarly journals Simultaneous Upregulation of Elastolytic and Elastogenic Factors Are Necessary for Regulated Collateral Diameter Expansion

2022 ◽  
Vol 8 ◽  
Author(s):  
Elizabeth Andraska ◽  
Nolan Skirtich ◽  
Dylan McCreary ◽  
Rohan Kulkarni ◽  
Edith Tzeng ◽  
...  
Keyword(s):  
Shear Stress ◽  
Elastic Fiber ◽  
Elastic Fibers ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Shear ◽  
High Shear Stress ◽  
Luminal Diameter ◽  
Time Points ◽  
Bonferroni Adjustment ◽  
Elastin Degradation

Background: During arteriogenesis, outward remodeling of the arterial wall expands luminal diameter to produce increased conductance in developing collaterals. We have previously shown that diameter expansion without loss of internal elastic lamina (IEL) integrity requires both degradation of elastic fibers and LOX-mediated repair. The aim of this study was to investigate the expression of genes involved in remodeling of the extracellular matrix (ECM) using a model of arteriogenesis.Methods: Sprague-Dawley rats underwent femoral artery ligation with distal arteriovenous fistula (FAL + AVF) placement. Profunda femoral arteries (PFA) were harvested for analysis at various time points. Serum desmosine, an amino acid found exclusively in elastin, was evaluated with enzyme-linked immunosorbent assay (ELISA) as a marker of tissue elastolysis. Tissue mRNA isolated from FAL + AVF exposed PFAs was compared to the contralateral sham-operated using qPCR. HCAECs were cultured under low shear stress (8 dyn·s/cm2) for 24 h and then exposed to high shear stress (40 dyn·s/cm2) for 2–6 h. Primers used included FBN-1, FBN-2, Timp-2, LOX-1, Trop-E, Cath-K, Cath-S, MMP-2, MMP-9, FBLN-4, and FBLN-5 and were normalized to GAPDH. mRNA fold changes were quantified using the 2-ΔΔCq method. Comparisons between time points were made with non-parametric ANOVA analysis with Bonferroni adjustment.Results: PFAs showed IEL reorganization during arteriogenesis. Serum desmosine levels are significantly elevated at 2 days and one week, with a return to baseline thereafter (p < 0.01). Expression of ECM structural proteins (FBN-1, FBN-2, FBLN-4, FBLN-5, Tropoelastin, TIMP-2, LOX-1) and elastolytic proteins (MMP-2, MMP-9, Cathepsin S, Cathepsin K) exhibited an early peak (p < 0.05) relative to sham PFAs. After two weeks, expression returned to baseline. HCAECs demonstrated upregulation of FBN-2, FBLN-5, LOX-1 and Trop-E at 4 h of high shear stress, as well as elastolytic protein MMP-2.Conclusions: Elastin degradation begins early in arteriogenesis and is mediated by local upregulation of elastolytic genes. Elastolysis appears to be simultaneously balanced by production of elastic fiber components which may facilitate stabilization of the IEL. Endothelial cells are central to initiation of arteriogenesis and begin ECM remodeling in response to altered shear stress.

ADAMTS13 Improving the Cell Engraftment Efficacy in Mouse Model of Bone Marrow Transplantation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1077-1077
Author(s):  
Hideto Matsui ◽  
Masaaki Doi ◽  
Yasunori Matsunari ◽  
Maiko Takeda ◽  
Kenji Nishio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Shear Stress ◽  
Cell Expansion ◽  
Essential Role ◽  
Donor Cell ◽  
High Shear ◽  
High Shear Stress ◽  
Wild Type ◽  
Cell Homing ◽  
Cell Engraftment

Abstract Abstract 1077 The adhesive protein von Willebrand factor (VWF) plays an essential role on physiologic hemostasis, mediating platelet aggregation under high shear stress conditions. However excessive functions of VWF could cause thrombotic occlusion of microvasculature such as arterial capillaries, where blood flow creates a typical high shear stress. The VWF-cleaving protease ADAMTS13 is therefore thought to down-regulate precisely the VWF function to maintain enriched microcirculation. In this context, we hypothesized that this ADAMTS13 role might contribute to better donor cell homing and engraftment in various cell therapy approaches, in which fluent blood flow could be critical in the microcirculation system. To test this hypothesis, we investigated the essential role of ADAMTS13 on the donor cell engraftment using bone marrow transplantation (BMT) model in Adamts13 −/− and wild-type mice. Irradiated recipient mice were received 2 × 106 GFP positive cells from the sex-matched GFP donor mice. All of irradiated recipient mice without receiving BMT died within 21 days. Although there is no difference between Adamts13 −/− and wild-type mice in survival rate after 7days of BMT, Kaplan-Meier analysis revealed that the percent ratio of survival rate starts significantly declining after 14 days of BMT in the group of Adamts13 −/− mice. The successful cell engraftment in BMT was assessed by the number of GFP-positive neutrophils in peripheral blood at the several time points from BMT. As a result, the duration achieving the number of GFP-positive neutrophils over 500/μL was found to significantly delay in the Adamts13 −/− mice, as compared with the wild-type mice (20.2±3.8 days vs. 14.4±3.3 days). However, histological examination during the whole observation periods could not detect any typical thrombotic lesions of micro vessels developed in both wild-type and Adamts13 −/− mice. In addition, the delayed cell engraftment observed in the Adamts13 −/− mice became normalized by the bolus administration of recombinant ADAMTS13 (10 μg/mouse) at the day 0 of BMT. Bone marrow analysis at the day 1 of BMT revealed that the number of GFP-positive blood cells in bone marrow was significantly reduced in the Adamts13 −/− mice as compared with the wild-type mice, which could result in the delayed cell expansion at the day 7 and day14 of BMT in Adamts13 −/− mice. The single bolus injection of recombinant ADAMTS13 was found to fully correct the delayed cell expansion in bone marrow in the Adamts13 −/− mice. Our results indicate that the regulation of VWF-mediated thrombotic or inflammatory responses by ADAMTS13 could contribute to better microcirculation which could be critical for efficient donor cell homing and engraftment in BMT, suggesting a therapeutic potential of ADAMTS13 in cell therapy approaches. Disclosures: Soejima: The Chemo-Sero-Therapeutic Research Institute: Employment.

Effects of Low ADAMTS 13 Levels and the Role of Von Willebrand Factor in Cardiovascular Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5320-5320
Author(s):  
Giorgio Corinaldesi
Keyword(s):  
Cardiovascular Disease ◽  
Shear Stress ◽  
Relative Risk ◽  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
High Shear ◽  
High Shear Stress ◽  
Increased Risk ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract 5320 Von Willebrand factor (vWF) is a large multimeric glycoprotein that plays an important role as a co-factor in platelet adhesion and aggregation and as the circulating carrier for factor VIII; increased vWF levels, and the persistence of unprocessed ultra-large vWF multimers (ULVWF) that are hyperactive to binding GPIb-IX-V complex with high-strength bonds, precipitate platelet clumping in arterioles and capillaries, causing platelet aggregation and thrombotic microangiopathy resulting in tissue ischemia with an increased risk of CAD and stroke. vWF is the major ligand binding the subunit of trombospondin, P-selectin, collagen and SLAM family (signaling lymphocytic activation molecule), mediates platelet translocation, tethering and rolling under high shear stress and adhesion. The activity of VWF is modulated by ADAMTS-13 a Zn2+/Ca2+ dependent metalloprotease that cleaves vWF to smaller and less active forms at the tyr842/met843 within the A2 domain, and cleaves the tyr 1605/met 1606; this mechanism reduces the molecular weight and platelet-tethering function of vWF to the sub endothelium at high shear stress. ADAMTS-13 levels also correlate positively with cholesterol, triglycerides and body mass index (BMI). In addition, we have observed a negative association with fibrinogen, C-reactive protein, and ion trasport K+dependent Na+/Ca2+ exchange (SLC24A3); the phosphorylation of tyrosine 1605 and threonine/serine kinase inhibitors have negative effects on integrin GPIIb-IIIa that showed decrease expression. In patients with a level of ADAMTS-13 below 38%, the presence of ultra large vWF multimers is high and the relative risk of ischemic heart disease was 18.2% (95% CI, 0.8 to 4.2), and the relative risk of stroke was 11.5 % (95% CI, 0.6 to 3.9). We investigated the relation between the low activity of ADAMTS-13 and AMI or stroke; we measured VWF and ADAMTS-13 antigen levels in 16 patients with AMI, in 10 patients with stroke, and in 16 control subjects. We demonstrated that vWF levels are significantly higher in patients with AMI or stroke, while a severe decrease of ADAMTS-13 activity augments the risk of cardiovascular disease, and cerebral ischemia; measurement of both VWF and ADAMTS-13 may provide a good indicator for the likelihood of AMI and stroke. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 5065Platelet desialylation induced by high shear-stress mechanical circulatory support

2018 ◽  
Vol 39 (suppl_1) ◽  
Author(s):  
H Spillemaeker ◽  
A Dupont ◽  
A Kauskot ◽  
A Rauch ◽  
F Vincent ◽  
...  
Keyword(s):  
Shear Stress ◽  
Mechanical Circulatory Support ◽  
Circulatory Support ◽  
High Shear ◽  
High Shear Stress

scholarly journals Nitric Oxide–Dependent and Nitric Oxide–Independent Transcriptional Responses to High Shear Stress in Endothelial Cells

Hypertension ◽  
2005 ◽  
Vol 45 (4) ◽  
pp. 672-680 ◽  
Author(s):  
Branko Braam ◽  
Remmert de Roos ◽  
Hans Bluyssen ◽  
Patrick Kemmeren ◽  
Frank Holstege ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
High Shear ◽  
High Shear Stress ◽  
Transcriptional Responses

DIFFUSION LIMITED AGGREGATION FROM SHEAR STRESS AS A SIMPLE MODEL OF VASCULOGENESIS

Fractals ◽  
1999 ◽  
Vol 07 (01) ◽  
pp. 33-39 ◽  
Author(s):  
VINCENT FLEURY ◽  
LAURENT SCHWARTZ
Keyword(s):  
Shear Stress ◽  
Dendritic Growth ◽  
High Shear ◽  
Vascular Pattern ◽  
Laplacian Growth ◽  
High Shear Stress ◽  
Diffusion Limited Aggregation ◽  
Diffusion Limited ◽  
Capillary Bed ◽  
Arteries And Veins

A model is proposed by which the formation of the vascular network in animals proceeds via progressive penetration of the vessel ramification into a capillary mesh, by means of a laplacian growth mechanism of hydrodynamical origin. In this model, the growth of both arteries and veins follows the directions of high shear stress provoked by the blood flow on the endothelial wall of a pre-existing capillary mesh. This process is shown to be identical to the phenomenon of dendritic growth, which is responsible for the formation of such well-known patterns as dendritic crystals, lightning sparks or branching aggregates of bacteria. A number of straightforward consequences of potentially important medical and physiological interests are deduced. These include the natural and spontaneous organization of the arterial and venal trees, the spontaneous and unavoidable tropism of arteries towards veins and vice-versa, the hierarchical character of the vessels and the possibility of computerized prediction of the vascular pattern from the shape of the capillary bed.

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