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Bernas M.J.,University of Arizona | Cardoso F.L.,University of Lisbon | Daley S.K.,University of Arizona | Weinand M.E.,University of Arizona | And 8 more authors.
Nature Protocols | Year: 2010

We describe a method for generating primary cultures of human brain microvascular endothelial cells (HBMVECs). HBMVECs are derived from microvessels isolated from temporal tissue removed during operative treatment of epilepsy. The tissue is mechanically fragmented and size filtered using polyester meshes. The resulting microvessel fragments are placed onto type I collagen-coated flasks to allow HBMVECs to migrate and proliferate. The overall process takes less than 3 h and does not require specialized equipment or enzymatic processes. HBMVECs are typically cultured for approximately 1 month until confluent. Cultures are highly pure (97% endothelial cells; 3% pericytes), are reproducible, and show characteristic brain endothelial markers (von Willebrand factor, glucose transporter-1) and robust expression of tight and adherens junction proteins as well as caveolin-1 and efflux protein P-glycoprotein. Monolayers of HBMVECs show characteristically high transendothelial electric resistance and have proven useful in multiple functional studies for in vitro modeling of the human blood-brain barrier. © 2010 Nature America, Inc. All rights reserved. Source


Junkin M.,University of Arizona | Lu Y.,University of Arizona | Long J.,University of Arizona | Deymier P.A.,University of Arizona | And 2 more authors.
Biomaterials | Year: 2013

Calcium signaling in the diverse vascular structures is regulated by a wide range of mechanical and biochemical factors to maintain essential physiological functions of the vasculature. To properly transmit information, the intercellular calcium communication mechanism must be robust against various conditions in the cellular microenvironment. Using plasma lithography geometric confinement, we investigate mechanically induced calcium wave propagation in networks of human umbilical vein endothelial cells organized. Endothelial cell networks with confined architectures were stimulated at the single cell level, including using capacitive force probes. Calcium wave propagation in the network was observed using fluorescence calcium imaging. We show that mechanically induced calcium signaling in the endothelial networks is dynamically regulated against a wide range of probing forces and repeated stimulations. The calcium wave is able to propagate consistently in various dimensions from monolayers to individual cell chains, and in different topologies from linear patterns to cell junctions. Our results reveal that calcium signaling provides a robust mechanism for cell-cell communication in networks of endothelial cells despite the diversity of the microenvironmental inputs and complexity of vascular structures. © 2012 Elsevier Ltd. Source


Gruionu G.,Harvard University | Hoying J.B.,Cardiovascular Innovation Institute | Pries A.R.,Charite - Medical University of Berlin | Secomb T.W.,University of Arizona
Microcirculation | Year: 2012

Objective: Vascular networks respond to chronic alterations in blood supply by structural remodeling. Previously, we showed that blood flow changes in the mouse GA lead to transient diameter increases, which can generate large increases in circumferential wall stress. Here, we examine the associated changes in the medial area of the arterial wall and the effects on circumferential wall stress. Methods: To induce blood flow changes, one of the two feeding vessels to the GA was surgically removed. At 7-56days after blood flow interruption, the vasculature was perfused with India ink for morphological measurements, and processed for immuno-cytochemistry to mark the medial cross-section area. Theoretical simulations of hemodynamics were used to analyze the data. Results: During adaptive increases in vessel diameter, increases in medial area were observed, most strongly in the middle region of the artery. Simulations showed that this increase in medial area limits the increase in estimated circumferential stress during vascular adaptation to less than 50%, in contrast to an increase of up to 250% if the medial area had remained unchanged. Conclusions: During vascular adaptation, increases in circumferential stress are limited by growth of the media coordinated with diameter changes. © 2012 John Wiley & Sons Ltd. Source


Krishnan L.,Cardiovascular Innovation Institute | Chang C.C.,Cardiovascular Innovation Institute | Nunes S.S.,Toronto General Research Institute | Williams S.K.,Cardiovascular Innovation Institute | And 2 more authors.
Critical Reviews in Biomedical Engineering | Year: 2013

The microvasculature is a dynamic cellular system necessary for tissue health and function. Therapeutic strategies that target the microvasculature are expanding and evolving, including those promoting angiogenesis and microvascular expansion. When considering how to manipulate angiogenesis, either as part of a tissue construction approach or a therapy to improve tissue blood flow, it is important to know the microenvironmental factors that regulate and direct neovessel sprouting and growth. Much is known concerning both diffusible and matrix-bound angiogenic factors, which stimulate and guide angiogenic activity. How the other aspects of the extravascular microenvironment, including tissue biomechanics and structure, influence new vessel formation is less well known. Recent research, however, is providing new insights into these mechanisms and demonstrating that the extent and character of angiogenesis (and the resulting new microcirculation) is significantly affected. These observations and the resulting implications with respect to tissue construction and microvascular therapy are addressed. © 2013 by Begell House, Inc. Source


Kondo K.,Emory University | Bhushan S.,Emory University | King A.L.,Emory University | Prabhu S.D.,University of Alabama at Birmingham | And 10 more authors.
Circulation | Year: 2013

Background-: Cystathionine γ-lyase (CSE) produces H2S via enzymatic conversion of L-cysteine and plays a critical role in cardiovascular homeostasis. We investigated the effects of genetic modulation of CSE and exogenous H2S therapy in the setting of pressure overload-induced heart failure. Methods and Results-: Transverse aortic constriction was performed in wild-type, CSE knockout, and cardiac-specific CSE transgenic mice. In addition, C57BL/6J or CSE knockout mice received a novel H2S donor (SG-1002). Mice were followed up for 12 weeks with echocardiography. We observed a >60% reduction in myocardial and circulating H2S levels after transverse aortic constriction. CSE knockout mice exhibited significantly greater cardiac dilatation and dysfunction than wild-type mice after transverse aortic constriction, and cardiac-specific CSE transgenic mice maintained cardiac structure and function after transverse aortic constriction. H2S therapy with SG-1002 resulted in cardioprotection during transverse aortic constriction via upregulation of the vascular endothelial growth factor-Akt-endothelial nitric oxide synthase-nitric oxide-cGMP pathway with preserved mitochondrial function, attenuated oxidative stress, and increased myocardial vascular density. Conclusions-: Our results demonstrate that H 2S levels are decreased in mice in the setting of heart failure. Moreover, CSE plays a critical role in the preservation of cardiac function in heart failure, and oral H2S therapy prevents the transition from compensated to decompensated heart failure in part via upregulation of endothelial nitric oxide synthase and increased nitric oxide bioavailability. © 2013 American Heart Association, Inc. Source

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