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Wang L.,U.S. National Institute of Standards and Technology | Bigos M.,Stanford University | Nolan J.P.,La Jolla Bioengineering Institute
Cytometry Part A | Year: 2012

Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue™), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned fluorescence standard beads. © 2012 International Society for Advancement of Cytometry. Source


Walshe T.E.,Schepens Eye Research Institute | Dela Paz N.G.,Schepens Eye Research Institute | Dela Paz N.G.,La Jolla Bioengineering Institute | D'Amore P.A.,Schepens Eye Research Institute
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2013

OBJECTIVE-: Vascular endothelial cells (ECs) are continuously exposed to blood flow that contributes to the maintenance of vessel structure and function; however, the effect of hemodynamic forces on transforming growth factor-β (TGF-β) signaling in the endothelium is poorly described. We examined the potential role of TGF-β signaling in mediating the protective effects of shear stress on ECs. APPROACH AND RESULTS-: Human umbilical vein ECs (HUVECs) exposed to shear stress were compared with cells grown under static conditions. Signaling through the TGF-β receptor ALK5 was inhibited with SB525334. Cells were examined for morphological changes and harvested for analysis by real-time polymerase chain reaction, Western blot analysis, apoptosis, proliferation, and immunocytochemistry. Shear stress resulted in ALK5-dependent alignment of HUVECs as well as attenuation of apoptosis and proliferation compared with static controls. Shear stress led to an ALK5-dependent increase in TGF-β3 and Krüppel-like factor 2, phosphorylation of endothelial NO synthase, and NO release. Addition of the NO donor S-nitroso-N- acetylpenicillamine rescued the cells from apoptosis attributable to ALK5 inhibition under shear stress. Knockdown of TGF-β3, but not TGF-β1, disrupted the HUVEC monolayer and prevented the induction of Krüppel-like factor 2 by shear. CONCLUSIONS-: Shear stress of HUVECs induces TGF-β3 signaling and subsequent activation of Krüppel-like factor 2 and NO, and represents a novel role for TGF-β3 in the maintenance of HUVEC homeostasis in a hemodynamic environment. © 2013 American Heart Association, Inc. Source


Patent
Yeshiva University and La Jolla Bioengineering Institute | Date: 2013-05-01

Nanoparticles are provided that comprise S-nitrosothiol (SNO) groups covalently bonded to the nanoparticles and/or S-nitrosothiol containing molecules encapsulated within the nanoparticles, as well as methods of making and using the nanoparticles. The invention also provides methods of preparing nanoparticles comprising Snitrosothiol (SNO) groups covalently bonded to the nanoparticles, where the methods comprise a) providing a buffer solution comprising chitosan, polyethylene glycol, nitrite, glucose, and hydrolyzed 3-mercaptopropyltrimethoxysilane (MPTS); b) adding hydrolyzed tetramethoxysilane (TMOS) to the buffer solution to produce a sol-gel; and c) lyophilizing and ball milling the sol-gel to produce nanoparticles of a desired size.


Melchior B.,La Jolla Bioengineering Institute | Frangos J.A.,La Jolla Bioengineering Institute
American Journal of Physiology - Cell Physiology | Year: 2010

Atheroprone regions of the arterial circulation are characterized by time-varying, reversing, and oscillatory wall shear stress. Several in vivo and in vitro studies have demonstrated that flow reversal (retrograde flow) is atherogenic and proinflammatory. The molecular and structural basis for the sensitivity of the endothelium to flow direction, however, has yet to be determined. It has been hypothesized that the ability to sense flow direction is dependent on the direction of inclination of the interendothelial junction. Immunostaining of the mouse aorta revealed an inclination of the cell-cell junction by 13° in direction of flow in the descending aorta where flow is unidirectional. In contrast, polygonal cells of the inner curvature where flow is disturbed did not have any preferential inclination. Using a membrane specific dye, the angle of inclination of the junction was dynamically monitored using live cell confocal microscopy in confluent human endothelial cell monolayers. Upon application of shear the junctions began inclining within minutes to a final angle of 10° in direction of flow. Retrograde flow led to a reversal of junctional inclination. Flow-induced junctional inclination was shown to be independent of the cytoskeleton or glycocalyx. Additionally, within seconds, retrograde flow led to significantly higher intracellular calcium responses than orthograde flow. Together, these results show for the first time that the endothelial intercellular junction inclination is dynamically responsive to flow direction and confers the ability to endothelial cells to rapidly sense and adapt to flow direction. Copyright © 2010 the American Physiological Society. Source


Melchior B.,La Jolla Bioengineering Institute | Frangos J.A.,La Jolla Bioengineering Institute
American Journal of Physiology - Cell Physiology | Year: 2012

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca 2+] i). Whether the source of [Ca 2+] i is extracellular, released from Ca 2+ intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca 2+] i burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα q/11) is a likely candidate in effecting shear-induced increases in [Ca 2+] i since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flowadapted human ECs, we have investigated to what extent the Gα q/11 pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca 2+] i response to a 10 dyn/cm 2 retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα q/11. A similar lag in [Ca 2+] i transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca 2+] i responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca 2+] i. Taken together, our results identify the Gα q/11-PLC pathway as the initial trigger for retrograde flowinduced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow. © 2012 the American Physiological Society. Source

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