Plasmatic and cell-based enhancement by microparticles originated from platelets and endothelial cells under simulated in vitro conditions of a dilutional coagulopathy

Abstract Background Aggressive fluid management and other external factors may lead to hypothermia, acidosis and hemodilution (defined as Lethal Triad, LT) contributing to a trauma-induced coagulopathy (TIC) that worsens patients’ outcomes. Procoagulant microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. However, their functions remain largely unexplored. This study aimed to characterize effects of MP subtypes and concentrations on functional coagulation under in vitro simulated conditions. Methods Blood from eleven volunteers were collected to simulate in vitro conditions of hemodilution (HD) and LT, respectively. HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. After introducing MP to in vitro conditions, we measured their concentration-dependent effects (1.000, 10.000 and 15.000 MP/μl blood) on coagulation compared to whole blood (WB). For each condition, coagulation was characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation using Thrombodynamics® technology. Results MP originated from platelets and endothelial cells affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities (10.000 and 15.000 PDMP/μl blood) significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions as well, only high PDMP concentration induced platelet activation, clot growth and size. In contrast, EDMP did not induce platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions. Discussion The study demonstrates an essential role of MP within the coagulation process under simulated coagulopathic conditions. PDMP affected platelets promoting clot formation likely by providing a surface enlargement. EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous clots. Conclusion Under simulated conditions of a dilutional coagulopathy, MP from different cellular origin indicate a divergent but both procoagulant mechanism within the coagulation process.

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Plasmatic and cell-based enhancement by microparticles originated from platelets and endothelial cells under simulated in vitro conditions of a dilutional coagulopathy

10.21203/rs.3.rs-49206/v3 ◽

2021 ◽

Author(s):

Julia Boehm ◽

Nadine Schäfer ◽

Marc Maegele ◽

Birgit Stümpges ◽

Ursula Bauerfeind ◽

...

Keyword(s):

Endothelial Cells ◽

Platelet Activation ◽

Fibrin Clot ◽

Dependent Manner ◽

Clotting Factors ◽

Coagulation Process ◽

Lethal Triad ◽

Dilutional Coagulopathy ◽

Plasmatic Coagulation

Abstract Background: Aggressive fluid management and other external factors may lead to hypothermia, acidosis and hemodilution (defined as Lethal Triad, LT) contributing to a trauma-induced coagulopathy (TIC) that worsens patients’ outcomes. Procoagulant microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. However, their functions remain largely unexplored. This study aimed to characterize effects of MP subtypes and concentrations on functional coagulation under in vitro simulated conditions. Methods: Blood from eleven volunteers were collected to simulate in vitro conditions of hemodilution (HD) and LT, respectively. HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. After introducing MP to in vitro conditions, we measured their concentration-dependent effects (1.000, 10.000 and 15.000 MP/µl blood) on coagulation compared to whole blood (WB). For each condition, coagulation was characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation using Thrombodynamics® technology.Results: MP originated from platelets and endothelial cells affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities (10.000 and 15.000 PDMP/µl blood) significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions as well, only high PDMP concentration induced platelet activation, clot growth and size. In contrast, EDMP did not induce platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions.Discussion: The study demonstrates an essential role of MP within the coagulation process under simulated coagulopathic conditions. PDMP affected platelets promoting clot formation likely by providing a surface enlargement. EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous clots.Conclusion: Under simulated conditions of a dilutional coagulopathy, MP from different cellular origin indicate a divergent but both procoagulant mechanism within the coagulation process.

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Plasmatic and cell-based enhancement of in vitro induced coagulopathy by microparticles originated from platelets and endothelial cells

10.21203/rs.3.rs-49206/v2 ◽

2020 ◽

Author(s):

Julia Boehm ◽

Nadine Schäfer ◽

Marc Maegele ◽

Birgit Stümpges ◽

Ursula Bauerfeind ◽

...

Keyword(s):

Endothelial Cells ◽

Platelet Activation ◽

Fibrin Clot ◽

Dependent Manner ◽

Clotting Factors ◽

Concentration Dependent Manner ◽

Coagulation Process ◽

Lethal Triad ◽

Plasmatic Coagulation

Abstract Background: Aggressive trauma management and other external factors lead to hypothermia, acidosis and hemodilution (defined as Lethal Triad, LT) contributing to coagulopathy after trauma (Trauma-induced coagulopathy, TIC) that worsens patients’ outcomes. Procoagulative microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. However, their functions remain largely unexplored. This study aimed to characterize effects of MP subtypes and concentrations on functional coagulation under in vitro simulated conditions. Methods: Blood from eleven volunteers were collected to simulate in vitro conditions of haemodilution (HD) and LT, respectively. HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. After introducing MP to in vitro conditions, their concentration-dependent effects (1.000, 10.000 and 15.000 MP/µl blood) on coagulation compared to whole blood (WB) were characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation and spontaneous clotting using Thrombodynamics® technology.Results: MP originated from platelets and endothelial cells affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions, the highest PDMP concentration enhanced platelet activation, clot growth and size. In contrast, EDMP supplementation did not affect platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions.Discussion: The study demonstrates an essential role of MP within the coagulation process under simulated coagulopathic conditions. While PDMP affected platelets promoting clot formation likely by providing a surface enlargement, EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous fibrin clots.Conclusion: The diverse effects of in vitro generated MP from different cellular origin indicate a divergent mechanism of action exhibiting distinct functions within the coagulation process.

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Divergent enhancement of in vitro induced coagulopathy by microparticle subtypes

10.21203/rs.3.rs-49206/v1 ◽

2020 ◽

Author(s):

Julia Boehm ◽

Nadine Schäfer ◽

Marc Maegele ◽

Birgit Stümpges ◽

Ursula Bauerfeind ◽

...

Keyword(s):

Cell Culture ◽

Platelet Activation ◽

Fibrin Clot ◽

Dependent Manner ◽

Clotting Factors ◽

Cellular Origin ◽

Coagulation Process ◽

Lethal Triad ◽

Plasmatic Coagulation

Abstract Background Aggressive fluid therapy of patients following a major trauma may result in hypothermia, acidosis and haemodilution that deteriorates even further towards a trauma-induced coagulopathy. The combination of these three factors is referred as lethal triad (LT) and a progressive course of LT is associated with a worsening or complete failure of haemodynamics and coagulative function. Within the process of coagulation, microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. This study aimed at the characterization of coagulative effects originating from MP with different cellular origin and concentration under in vitro simulated traumatic conditions of haemodilution (HD) and LT. Methods HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. Effects of MP on coagulation depending on concentrations (1.000, 10.000 and 15.000 MP/µl blood) were characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation and spontaneous clotting using Thrombodynamics® technology. Results MP originated from platelets and endothelial cell culture affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions, the highest PDMP concentration enhanced platelet activation, clot growth and size. In contrast, EDMP supplementation did not affect platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions. Discussion The study demonstrates the essential role of MP within the coagulation process in the simulated traumatic conditions. While PDMP affected platelets promoting clot formation likely by providing a surface enlargement, EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous fibrin clots. Conclusion The diverse effects of in vitro generated MP from different cellular origin indicate a divergent mechanism of action exhibiting distinct functions within the coagulation process.

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Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.1.85 ◽

2001 ◽

Vol 12 (1) ◽

pp. 85-100 ◽

Cited By ~ 111

Author(s):

Meredith Gonzales ◽

Babette Weksler ◽

Daisuke Tsuruta ◽

Robert D. Goldman ◽

Kristine J. Yoon ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factors ◽

Endothelial Cell ◽

Branching Morphogenesis ◽

Basement Membranes ◽

Αvβ3 Integrin ◽

Dependent Manner ◽

Matrix Adhesion ◽

Focal Contacts

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

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Effect of SR121566A, a Potent GP IIb-IIIa Antagonist, on the HIT Serum/heparin-Induced Platelet Mediated Activation of Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615196 ◽

1998 ◽

Vol 80 (08) ◽

pp. 326-331 ◽

Cited By ~ 25

Author(s):

Pierre Savi ◽

Walter Jeske ◽

Jeanine Walenga ◽

Jean-Marc Herbert

Keyword(s):

Endothelial Cells ◽

Platelet Aggregation ◽

Umbilical Vein ◽

Common Adverse Effect ◽

Surface Protein ◽

Heparin Therapy ◽

Serotonin Release ◽

Dependent Manner ◽

Platelet Factor

SummaryHeparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1β, IL6, TNFα and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-IIIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPγS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPγS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs.Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.

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Kisspeptin-10 Inhibits Angiogenesis in Human Placental Vessels ex Vivo and Endothelial Cells in Vitro

Endocrinology ◽

10.1210/en.2010-0565 ◽

2010 ◽

Vol 151 (12) ◽

pp. 5927-5934 ◽

Cited By ~ 32

Author(s):

Thayalini Ramaesh ◽

James J. Logie ◽

Antonia K. Roseweir ◽

Robert P. Millar ◽

Brian R. Walker ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Vessels ◽

Ex Vivo ◽

Umbilical Vein ◽

Biologically Active ◽

Trophoblast Invasion ◽

In Vitro Assays ◽

Dependent Manner ◽

Inhibition Of Proliferation

Recent studies suggest that kisspeptin (a neuropeptide central to the regulation of gonadotrophin secretion) has diverse roles in human physiology, including a putative role in implantation and placental function. Kisspeptin and its receptor are present in human blood vessels, where they mediate vasoconstriction, and kisspeptin is known to inhibit tumor metastasis and trophoblast invasion, both processes involving angiogenesis. We hypothesized that kisspeptin contributes to the regulation of angiogenesis in the reproductive system. The presence of the kisspeptin receptor was confirmed in human placental blood vessels and human umbilical vein endothelial cells (HUVEC) using immunochemistry. The ability of kisspeptin-10 (KP-10) (a shorter biologically active processed peptide) to inhibit angiogenesis was tested in explanted human placental arteries and HUVEC using complementary ex vivo and in vitro assays. KP-10 inhibited new vessel sprouting from placental arteries embedded in Matrigel and tube-like structure formation by HUVEC, in a concentration-dependent manner. KP-10 had no effect on HUVEC viability or apoptosis but induced concentration-dependent inhibition of proliferation and migration. In conclusion, KP-10 has antiangiogenic effects and, given its high expression in the placenta, may contribute to the regulation of angiogenesis in this tissue.

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Membrane Estrogen Receptor-Dependent Extracellular Signal-Regulated Kinase Pathway Mediates Acute Activation of Endothelial Nitric Oxide Synthase by Estrogen in Uterine Artery Endothelial Cells

Endocrinology ◽

10.1210/en.2003-0547 ◽

2004 ◽

Vol 145 (1) ◽

pp. 113-125 ◽

Cited By ~ 124

Author(s):

Dong-bao Chen ◽

Ian M. Bird ◽

Jing Zheng ◽

Ronald R. Magness

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Estrogen Receptor ◽

Plasma Membrane ◽

Uterine Artery ◽

Fluorescein Isothiocyanate ◽

Estrogen Receptor Α ◽

Dependent Manner ◽

Enos Phosphorylation

Abstract Rapid uterine vasodilatation after estrogen administration is believed to be mediated by endothelial production of nitric oxide (NO) via endothelial NO synthase (eNOS). However, the mechanism(s) by which estrogen activates eNOS in uterine artery endothelial cells (UAEC) is unknown. In this study, we observed that estradiol-17β (E2) and E2-BSA rapidly (<2 min) increased total NOx production in UAEC in vitro. This was associated with rapid eNOS phosphorylation and activation but was unaltered by pretreatment with actinomycin-D. estrogen receptor-α protein was detectable in isolated plasma membrane proteins by immunoblotting, and E2-BSA-fluorescein isothiocyanate binding was evident on the plasma membrane of UAEC. E2 did not mobilize intracellular Ca2+, but E2 and ionomycin in combination induced greater eNOS phosphorylation than either E2 or ionomycin alone. E2 did not stimulate rapid Akt phosphorylation. E2 stimulated rapid ERK2/1 activation in a time- and dose-dependent manner, with maximal responses observed at 5–10 min with E2 (10 nm to 1 μm) treatment. Acute activation of eNOS and NOx production by E2 could be inhibited by PD98059 but not by LY294002. When E2-BSA was applied, similar responses in NOx production, eNOS, and ERK2/1 activation to those of E2 were achieved. In addition, E2 and E2-BSA-induced ERK2/1 activation and ICI 182,780 could inhibit NOx production by E2. Thus, acute activation of eNOS to produce NO in UAEC by estrogen is at least partially through an ERK pathway, possibly via estrogen receptor localized on the plasma membrane. This pathway may provide a novel mechanism for NO-mediated rapid uterine vasodilatation by estrogen.

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Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation

International Journal of Molecular Sciences ◽

10.3390/ijms19092753 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2753 ◽

Cited By ~ 6

Author(s):

Nina Zippel ◽

Annemarieke Loot ◽

Heike Stingl ◽

Voahanginirina Randriamboavonjy ◽

Ingrid Fleming ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular Reactivity ◽

Umbilical Vein ◽

No Production ◽

Dependent Manner ◽

Calmodulin Binding ◽

Umbilical Vein Endothelial Cells ◽

Endothelial Nitric Oxide ◽

Specific Deletion

AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα−/−) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.

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Shear Stress Modulates the Expression of Thrombospondin-1 and CD36 in Endothelial Cells in vitro and during Shear Stress-Induced Angiogenesis in vivo

International Journal of Immunopathology and Pharmacology ◽

10.1177/205873920601900104 ◽

2006 ◽

Vol 19 (1) ◽

pp. 205873920601900 ◽

Cited By ~ 35

Author(s):

M. Bongrazio ◽

L. DA Silva-Azevedo ◽

E.C. Bergmann ◽

O. Baum ◽

B. Hinz ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Western Blot ◽

Dependent Manner ◽

Vascular Networks ◽

Thrombospondin 1 ◽

Matrix Bound ◽

Stress Dependent

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/1 drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress down-regulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

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Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Circulation ◽

10.1161/circ.118.suppl_18.s_576-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Bin Ren ◽

Arpita Mukhopadhyay* ◽

Anthony A Lanahan ◽

Zhen W Zhuang ◽

Karen L Moodie ◽

...

Keyword(s):

Endothelial Cells ◽

Branching Morphogenesis ◽

Erk Signaling ◽

Dependent Manner ◽

Wild Type ◽

Erk Activation ◽

Arterial Development ◽

Constitutively Active

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

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