scholarly journals An Ex Vivo Vessel Injury Model to Study Remodeling

2018 ◽  
Vol 27 (9) ◽  
pp. 1375-1389 ◽  
Author(s):  
Mehmet H. Kural ◽  
Guohao Dai ◽  
Laura E. Niklason ◽  
Liqiong Gui
Keyword(s):  
Shear Stress ◽  
Animal Models ◽  
Intimal Hyperplasia ◽  
Vascular Remodeling ◽  
Ex Vivo ◽  
Vessel Injury ◽  
Injury Model ◽  
Human Arteries

Objective: Invasive coronary interventions can fail due to intimal hyperplasia and restenosis. Endothelial cell (EC) seeding to the vessel lumen, accelerating re-endothelialization, or local release of mTOR pathway inhibitors have helped reduce intimal hyperplasia after vessel injury. While animal models are powerful tools, they are complex and expensive, and not always reflective of human physiology. Therefore, we developed an in vitro 3D vascular model validating previous in vivo animal models and utilizing isolated human arteries to study vascular remodeling after injury. Approach: We utilized a bioreactor that enables the control of intramural pressure and shear stress in vessel conduits to investigate the vascular response in both rat and human arteries to intraluminal injury. Results: Culturing rat aorta segments in vitro, we show that vigorous removal of luminal ECs results in vessel injury, causing medial proliferation by Day-4 and neointima formation, with the observation of SCA1+ cells (stem cell antigen-1) in the intima by Day-7, in the absence of flow. Conversely, when endothelial-denuded rat aortae and human umbilical arteries were subjected to arterial shear stress, pre-seeding with human umbilical ECs decreased the number and proliferation of smooth muscle cell (SMC) significantly in the media of both rat and human vessels. Conclusion: Our bioreactor system provides a novel platform for correlating ex vivo findings with vascular outcomes in vivo. The present in vitro human arterial injury model can be helpful in the study of EC-SMC interactions and vascular remodeling, by allowing for the separation of mechanical, cellular, and soluble factors.

Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodeling

2021 ◽  
Author(s):  
Lingfeng Luo ◽  
Yishuai Zhang ◽  
Chia Hsu ◽  
Vyacheslav A Korshunov ◽  
Xiaochun Long ◽  
...  
Keyword(s):  
Intimal Hyperplasia ◽  
Vascular Remodeling ◽  
Drug Target ◽  
Ex Vivo ◽  
Intimal Thickening ◽  
Therapeutic Approaches ◽  
Drug Eluting

Abstract Aims Intimal hyperplasia is a common feature of vascular remodeling disorders. Accumulation of synthetic smooth muscle cell (SMC)-like cells is the main underlying cause. Current therapeutic approaches including drug-eluting stents are not perfect due to the toxicity on endothelial cells and novel therapeutic strategies are needed. Our preliminary screening for dysregulated cyclic nucleotide phosphodiesterases (PDEs) in growing SMCs revealed the alteration of PDE10A expression. Herein, we investigated the function of PDE10A in SMC proliferation and intimal hyperplasia both in vitro and in vivo. Methods and results RT-qPCR, immunoblot, and in situ proximity ligation assay were performed to determine PDE10A expression in synthetic SMCs and injured vessels. We found that PDE10A mRNA and/or protein levels are up-regulated in cultured SMCs upon growth stimulation, as well as in intimal cells in injured mouse femoral arteries. To determine the cellular functions of PDE10A, we focused on its role in SMC proliferation. The anti-mitogenic effects of PDE10A on SMCs were evaluated via cell counting, BrdU incorporation, and flow cytometry. We found that PDE10A deficiency or inhibition arrested the SMC cell cycle at G1-phase with a reduction of cyclin D1. The anti-mitotic effect of PDE10A inhibition was dependent on cGMP-dependent protein kinase Iα (PKGIα), involving C-natriuretic peptide (CNP) and particulate guanylate cyclase natriuretic peptide receptor 2 (NPR2). In addition, the effects of genetic depletion and pharmacological inhibition of PDE10A on neointimal formation were examined in a mouse model of femoral artery wire injury. Both PDE10A knockout and inhibition decreased injury-induced intimal thickening in femoral arteries by at least 50%. Moreover, PDE10A inhibition decreased ex vivo remodeling of cultured human saphenous vein segments. Conclusions Our findings indicate that PDE10A contributes to SMC proliferation and intimal hyperplasia at least partially via antagonizing CNP/NPR2/cGMP/PKG1α signaling, and suggest that PDE10A may be a novel drug target for treating vascular occlusive disease. Translational perspective Coronary artery disease is currently the leading cause of death worldwide. SMCs are a major contributor to angioplasty restenosis, graft stenosis, and accelerated atherosclerosis. Current therapeutic approaches including drug-eluting stents targeting cell growth still have limitations. By combining studies on cultured SMCs in vitro, animal surgical models in vivo, and a human organ culture model ex vivo, we revealed an important role of PDE10A in modulating SMC proliferation and injury-induced intimal thickening. Given that PDE10A has been proven to be a safe drug target, its inhibition may represent a novel therapeutic strategy for vascular diseases associated with intimal hyperplasia.

An Innovative Ex Vivo Vascular Bioreactor as Comprehensive Tool to Study the Behavior of Native Blood Vessels Under Physiologically Relevant Conditions

2019 ◽  
Vol 2 (4) ◽  
Author(s):  
Noemi Vanerio ◽  
Marco Stijnen ◽  
Bas A. J. M. de Mol ◽  
Linda M. Kock
Keyword(s):  
Shear Stress ◽  
Blood Vessels ◽  
Ultrasound Imaging ◽  
Ex Vivo ◽  
Biological Response ◽  
Vessel Diameter ◽  
Tissue Growth ◽  
In Vivo Models

Abstract Ex vivo systems represent important models to study vascular biology and to test medical devices, combining the advantages of in vitro and in vivo models such as controllability of parameters and the presence of biological response, respectively. The aim of this study was to develop a comprehensive ex vivo vascular bioreactor to long-term culture and study the behavior of native blood vessels under physiologically relevant conditions. The system was designed to allow for physiological mechanical loading in terms of pulsatile hemodynamics, shear stress, and longitudinal prestretch and ultrasound imaging for vessel diameter and morphology evaluation. In this first experience, porcine carotid arteries (n = 4) from slaughterhouse animals were cultured in the platform for 10 days at physiological temperature, CO2 and humidity using medium with blood-mimicking viscosity, components, and stability of composition. As expected, a significant increase in vessel diameter was observed during culture. Flow rate was adjusted according to diameter values to reproduce and maintain physiological shear stress, while pressure was kept physiological. Ultrasound imaging showed that the morphology and structure of cultured arteries were comparable to in vivo. Histological analyses showed preserved endothelium and extracellular matrix and neointimal tissue growth over 10 days of culture. In conclusion, we have developed a comprehensive pulsatile system in which a native blood vessel can be cultured under physiological conditions. The present model represents a significant step toward ex vivo testing of vascular therapies, devices, drug interaction, and as basis for further model developments.

Autonomous Effects of Shear Stress and Cyclic Circumferential Stretch Regarding Endothelial Dysfunction and Oxidative Stress: An Ex Vivo Arterial Model

2009 ◽  
Author(s):  
Tyler Thacher ◽  
Rafaela da Silva ◽  
Paolo Silacci ◽  
Nikos Stergiopulos
Keyword(s):  
Shear Stress ◽  
Endothelial Dysfunction ◽  
Ex Vivo ◽  
Cyclic Stretch ◽  
Arterial Model ◽  
Individual Contributions ◽  
Independent Effects ◽  
And Oxidative Stress

Within the vasculature endothelial cells are constantly exposed to dynamic mechanical forces generated by pulsatile blood flow. Two stimuli known to modulate endothelial function are shear stress and cyclic circumferential strain. Yet, in most studies these two stimuli are simultaneously coupled in-vivo, making it very difficult to understand their individual contributions to vascular disease. Some attempts have been made to de-couple stretch and shear stress in-vitro by using different cell lines in a variety of stretch systems and flow chambers, straying from reality and making it hard to draw definitive conclusions. In this study we wish to find a compromise between the in-vivo and in-vitro work of the past by studying the independent effects of shear stress and cyclic stretch and how they contribute to endothelial dysfunction.

scholarly journals A Novel Platelet-Targeting Fibrinolytic Strategy: Endocytosed Urokinase By Megakaryocytes Is Stored in α-Granules and Is Functional In Vivo in a Murine Model of Thrombosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3627-3627
Author(s):  
Hyunsook Ahn ◽  
John Tkaczynski ◽  
Khalil Bdeir ◽  
Victoria Stepanova ◽  
Douglas B. Cines ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Point Of Care ◽  
Ldl Receptor ◽  
Single Amino Acid ◽  
Platelet Glycoprotein ◽  
Single Chain ◽  
Injury Model

We have been interested in developing fibrinolytic agents that have a prolonged half-life and can selectively prevent new thrombi from forming without destabilizing established hemostatic clots. We believe that platelet-targeting of urokinase plasminogen activator (uPA) might be able to achieve this goal. Two approaches had been tried in the past: In the first, we had generated chimeric drugs consisting of a platelet-targeting scFvs fused to a thrombin activatable low molecular weight (LMW) uPA (uPA-T). Infused scFv/uPA-T bound to circulating platelets that targeted the fusion within nascent thrombi where significant amounts of thrombin are generated to activate surface-bound scFv/uPA-T. In contrast, mature thrombi are spared because scFv/uPA-T platelets are not activated on the "shell", nor do they penetrate the "core" where thrombin might be present. Murine studies affirmed αIIbβ3-directed scFv/uPA-T provided thromboprophylaxis, but therapeutic doses caused significant thrombocytopenia in murine and baboon models. We therefore considered a second approach: ectopic storage of uPA during megakaryopoiesis. We found that scuPA was stored in platelet α-granules of transgenic mice that ectopically express single-chain uPA (scuPA) and did not cause systemic fibrinolysis. Infusion of such "scuPA platelets" into wildtype mice was highly effective at preventing new thrombi from developing. We now wish to develop this strategy further by taking advantage of ongoing efforts by others to generate in vitro-grown megakaryocytes (Mks) and platelets that can be modified to express uPA near the point-of-care and then infused into patients, bypassing the need to establish uPA-expressing hematopoietic cell lines. We have previously shown that Mks express low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) during their maturation, whereas the platelets that are released do not. We now asked whether in vitro-grown Mks beginning with CD34+ hematopoietic cells would endocytose uPA as seen in other cell types. We show that Mks internalize and store scuPA (scu-Mks, Fig. 1A), as well as LMW uPA and uPA-T (not shown) after overnight incubation. Endocytosed uPA is found within membrane bound structures that partly colocalize with von Willebrand factor (VWF)-positive granules, suggesting uPA is sorted to α-granules (Fig. 1A). Uptake is blocked by receptor-associated protein (RAP), which inhibits endocytosis by LDL receptor family members, including LRP1. We then studied whether platelets with endocytosed scuPA (scuPA-Plts) prevent nascent thrombus development in immunodeficient NOD-scid IL2rγnull (NSG) mice that are also homozygous for VWFR1326H (a single amino acid substitution that switches species selectivity of VWF so that it binds human platelet glycoprotein (GP) Ib/IX receptor rather than mouse GPIb/IX). These mice show a mild bleeding diathesis in a Rose Bengal photochemical carotid artery injury model unless they are infused with human Mks, which we have shown go on to release functional platelets in the recipient animal in its pulmonary capillary bed over the ensuing several hours (Fig. 1B). Thus, when 106 Mks not exposed to uPA were infused so that ~1-10% of circulating platelets were human, thrombi developed in this model; however similar infusion of scuPA-Mks did not occlude (Fig. 1B). In tail clip studies in the same genotypic mice, where the mice were first corrected with human platelets (Fig. 1C) followed 10 min later by scuPA-Mks, rebleeding did not develop when given a similar dose of scuPA-Mks that prevented thrombosis in the photochemical injury model. These studies suggest that Mks internalize biologically relevant concentrations of uPA through a process likely to involve LRP1. Whether ex vivo loading of Mks with uPA can serve as model for point-of-care therapeutics for thromboprophylaxis and diverse other hematologic and non-hematologic indications should be explored. Disclosures No relevant conflicts of interest to declare.

Imaging fibrin formation and platelet and endothelial cell activation in vivo

2011 ◽  
Vol 105 (05) ◽  
pp. 776-782 ◽  
Author(s):  
Bruce Furie ◽  
Lola Bellido-Martin ◽  
Vivien Chen ◽  
Reema Jasuja ◽  
Barbara Furie
Keyword(s):  
Blood Flow ◽  
Endothelial Cell ◽  
Animal Models ◽  
Cell Activation ◽  
In Vitro Assays ◽  
Endothelial Cell Activation ◽  
The Past ◽  
Injury Model

SummaryOver the past six decades research employing in vitro assays has identified enzymes, cofactors, cell receptors and associated ligands important to the haemostatic process and its regulation. These studies have greatly advanced our understanding of the molecular and cellular bases of haemostasis and thrombosis. However, in vitro assays cannot simultaneously reproduce the interactions of all of the components of the haemostatic process that occur in vivo nor do they reflect the importance of haemodynamic factors resulting from blood flow. To overcome these limitations investigators have increasingly turned to animal models of haemostasis and thrombosis. In this article we describe some advances in the visualisation of platelet and endothelial cell activation and blood coagulation in vivo and review what we have learned from our intravital microscopy experiments using primarily the laser-induced injury model for thrombosis.

scholarly journals Atherosclerosis and Thrombosis: Insights from Large Animal Models

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Gemma Vilahur ◽  
Teresa Padro ◽  
Lina Badimon
Keyword(s):  
Animal Models ◽  
Ex Vivo ◽  
Large Animal ◽  
Isolated Cells ◽  
Large Animal Models ◽  
Thrombosis Research ◽  
Human Pathology ◽  
Thrombotic Complications

Atherosclerosis and its thrombotic complications are responsible for remarkably high numbers of deaths. The combination ofin vitro, ex vivo, andin vivoexperimental approaches has largely contributed to a better understanding of the mechanisms underlying the atherothrombotic process. Indeed, different animal models have been implemented in atherosclerosis and thrombosis research in order to provide new insights into the mechanisms that have already been outlined in isolated cells and protein studies. Yet, although no model completely mimics the human pathology, large animal models have demonstrated better suitability for translation to humans. Indeed, direct translation from mice to humans should be taken with caution because of the well-reported species-related differences. This paper provides an overview of the availableatherothrombotic-likeanimal models, with a particular focus on large animal models of thrombosis and atherosclerosis, and examines their applicability for translational research purposes as well as highlights species-related differences with humans.

scholarly journals Retinal Organ Cultures as Alternative Research Models

2019 ◽  
Vol 47 (1) ◽  
pp. 19-29 ◽  
Author(s):  
Sven Schnichels ◽  
Tobias Kiebler ◽  
José Hurst ◽  
Ana M. Maliha ◽  
Marina Löscher ◽  
...  
Keyword(s):  
Animal Models ◽  
Organ Culture ◽  
Ex Vivo ◽  
Organ Cultures ◽  
Culture Systems ◽  
Cellular Processes ◽  
Experimental Systems ◽  
Vessel Morphology

Ex vivo organ cultures represent unique research models, as they combine the advantages of cell cultures with those of animal models. Being able to mimic in vivo situations through the use of organ cultures provides an excellent opportunity to investigate cellular processes, molecular pathways and cell–cell interactions, as well as structural and synaptic organisation. Human and animal organ cultures are now well established and comprise sensitive, easy-to-manipulate experimental systems that raise minimal ethical concerns. The eye, in particular, is a very complex organ that is not easy to reproduce in vitro. However, a lot of research has been dedicated to the development of suitable ocular organ cultures. This review covers the various ex vivo retinal organ culture systems available for use in ophthalmology research and compares them with commonly used animal models. In particular, bovine and porcine retinal organ culture systems are described, because the size, anatomy, physiology and vessel morphology of bovine and porcine eyes are similar to the human eye in an undisputed way, thus making them good models. In addition, these animals are widely used by the food industry and the eyes are considered surplus material. A short overview of murine, rat, rabbit, cat, canine and simian retinal organ cultures is also provided.

scholarly journals Research Models for Studying Vascular Calcification

2020 ◽  
Vol 21 (6) ◽  
pp. 2204 ◽  
Author(s):  
Jaqueline Herrmann ◽  
Milen Babic ◽  
Markus Tölle ◽  
Markus van der Giet ◽  
Mirjam Schuchardt
Keyword(s):  
Animal Models ◽  
Vascular Calcification ◽  
Ex Vivo ◽  
Systemic Disease ◽  
Treatment Options ◽  
Aortic Tissue ◽  
Cardiovascular Morbidity And Mortality ◽  
Culture Models

Calcification of the vessel wall contributes to high cardiovascular morbidity and mortality. Vascular calcification (VC) is a systemic disease with multifaceted contributing and inhibiting factors in an actively regulated process. The exact underlying mechanisms are not fully elucidated and reliable treatment options are lacking. Due to the complex pathophysiology, various research models exist evaluating different aspects of VC. This review aims to give an overview of the cell and animal models used so far to study the molecular processes of VC. Here, in vitro cell culture models of different origins, ex vivo settings using aortic tissue and various in vivo disease-induced animal models are summarized. They reflect different aspects and depict the (patho)physiologic mechanisms within the VC process.

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