On-Chip Endothelial inflammatorisk Fenotypning
Summary
Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.
Abstract
Aterogenes potentieras av metabola störningar som bidrar till en ökad tillstånd av systemisk inflammation resulterar i endotel dysfunktion. Dock tidiga funktionella förändringar i endotelet som betyder en individs risknivå inte direkt bedömas kliniskt för att vägleda terapeutisk strategi. Dessutom bidrar regleringen av inflammation av lokala hemodynamiken till den icke-slumpmässiga geografiska fördelningen av åderförkalkning, men mekanismerna är svåra att avgränsa in vivo. Vi beskriver ett lab-on-a-chip metod för att kvantitativt analys metabolisk störning av inflammatoriska händelser i humana endotelceller (EG) och monocyter enligt exakta flöde. Standardmetoder för mjuk litografi används för att microfabricate vaskulära mimetiska mikrofluidiska kammare (VMMC), som är bundna direkt till odlade EC monoskikt. 1 Dessa anordningar har fördelen med att använda små volymer av reagens medantillhandahålla en plattform för direkt avbildning av inflammatoriska händelser på membranet i EG utsätts för en väldefinierad skjuvning området. Vi har framgångsrikt tillämpat dessa enheter för att undersöka cytokin-, 2 lipid-3, 4 och RAGE-inducerad 5 inflammation i mänsklig aorta EG (HAEC). Här har vi dokumentera användningen av VMMC att analysera monocytisk cell (THP-1) valsning och gripande om HAEC monolager som konditioneras under egenskaper differentiella skjuv-och aktiveras av inflammatoriska cytokin TNF-α. Studier som dessa tillhandahåller mekanistiska inblick i atherosusceptibility i metabola riskfaktorer.
Protocol
Discussion
We describe the use of microfluidic PDMS devices for the on-chip assessment of endothelial inflammatory phenotype through the real-time imaging of CAM expression and monocyte adhesion. A major advantage of our approach lies in the ability to quantify outcomes associated with endothelial dysfunction in cells exposed to inflammatory mediators such as dietary lipids and cytokines under defined hydrodynamic conditions that mirror shear stress in atherogenic vessels. The VMMC facilitate the use of small amounts of reagents or…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH/NHLBI grant R01 HL082689 to Scott I. Simon and Anthony G. Passerini.
Materials
| Item | Company | Catalogue Number |
| 100 x 20mm Petri Dishes | BD Falcon | 353003 |
| Ethanol 95% | EMD Chemicals | EX0290-1 |
| DPBS | Cellgro | 21-031-CV |
| Type I Rat Tail Derived Collagen | Gibco | A10483-01 |
| Human Aortic Endothelial Cells | Genlantis | PH30405A |
| Antibiotic-Antimycotic Solution | Invitrogen | 15240-062 |
| Endothelial BulletKit | Lonza | CC-4176 |
| Endothelial Basal Media-2 | Lonza | CC-3156 |
| 10 ml Syringes | BD Falcon | 309604 |
| Polyurethane tubing | Tygon | ABW0001 |
| Leibovitz-15 Media | Gibco | 11415-069 |
| Sylgard 184 Silicone Elastomer Base | Dow Corning | 184 |
| Sylgard 184 Silicone Elastomer Curing Agent | Dow Corning | 184 |
| SU8 Photoresist Master Wafer | UC Davis Pan Lab | N/A |
| Eclipse TE200 Inverted Microscope | Nikon | Eclipse TE200 |
| Syringe Pump | Harvard Apparatus | PHD2000 |
| 19 gauge hypodermic needle | Kendall | 8881 |
| THP-1 Monocytic Cell Line | ATCC | TIB-202 |
| HBSS (Hanks Buffered Saline Solution) with Ca2+/Mg2+ | Gibco | 14025-092 |
| Tumor Necrosis Factor Alpha (TNF-α) | R&D Systems | 210-TA-010 |
| Stromal Derived Factor – 1 (SDF-1) | R&D Systems | 350-NS-010 |
| RPMI 1640 | Cellgro | 10-040-CV |
| Human Serum Albumin (HSA) | ZLB Behring | NDC 0053-7680-32 |
Table 2. Specific reagents and equipment.
Riferimenti
- Schaff, U. Y., Xing, M. M., Lin, K. K., Pan, N., Jeon, N. L., Simon, S. I. Vascular mimetics based on microfluidics for imaging the leukocyte–endothelial inflammatory response. Lab Chip. 7, 448-456 (2007).
- Tsou, J. K., Gower, R. M., Ting, H. J., Schaff, U. Y., Insana, M. F., Passerini, A. G., Simon, S. I. Spatial regulation of inflammation by human aortic endothelial cells in a linear gradient of shear stress. Microcirculation. 15, 311-323 (2008).
- Ting, H. J., Stice, J. P., Schaff, U. Y., Hui, D. Y., Rutledge, J. C., Knowlton, A. A., Passerini, A. G., Simon, S. I. Triglyceride-rich lipoproteins prime aortic endothelium for an enhanced inflammatory response to tumor necrosis factor-alpha. Circ. Res. 100, 381-390 (2007).
- Wang, Y. I., Schulze, J., Raymond, N., Tomita, T., Tam, K., Simon, S. I., Passerini, A. G. Endothelial inflammation correlates with subject triglycerides and waist size after a high-fat meal. Am. J. Physiol. Heart Circ. Physiol. 300, H784-H791 (2011).
- Deverse, J. S., Bailey, K. A., Jackson, K. N., Passerini, A. G. Shear stress modulates rage-mediated inflammation in a model of diabetes-induced metabolic stress. Am. J. Physiol. Heart Circ. Physiol. , (2012).
- Dai, G., Kaazempur-Mofrad, M. R., Natarajan, S., Zhang, Y., Vaughn, S., Blackman, B. R., Kamm, R. D., Garcia-Cardena, G., Gimbrone, M. A. Distinct endothelial phenotypes evoked by arterial waveforms derived from atherosclerosis-susceptible and -resistant regions of human vasculature. Proc. Natl. Acad. Sci. U.S.A. 101, 14871-14876 (2004).
- Young, E. W., Wheeler, A. R., Simmons, C. A. Matrix-dependent adhesion of vascular and valvular endothelial cells in microfluidic channels. Lab Chip. 7, 1759-1766 (2007).
- Davies, P. F., Civelek, M., Fang, Y., Guerraty, M. A., Passerini, A. G. Endothelial heterogeneity associated with regional athero-susceptibility and adaptation to disturbed blood flow in vivo. Semin Thromb Hemost. 36, 265-275 (2008).
- Burdge, G. C., Calder, P. C. Plasma cytokine response during the postprandial period: A potential causal process in vascular disease. Br. J. Nutr. 93, 3-9 (2005).
- Hotamisligil, G. S. Inflammation and metabolic disorders. Nature. 444, 860-867 (2006).
- Libby, P. Inflammation in atherosclerosis. Nature. 420, 868-874 (2002).
- Nigro, P., Abe, J., Berk, B. C. Flow shear stress and atherosclerosis: A matter of site specificity. Antioxid Redox Signal. 15, 1405-1414 (2011).
- Chiu, J. J., Lee, P. L., Chen, C. N., Lee, C. I., Chang, S. F., Chen, L. J., Lien, S. C., Ko, Y. C., Usami, S., Chien, S. Shear stress increases icam-1 and decreases vcam-1 and e-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells. Arterioscler. Thromb. Vasc. Biol. 24, 73-79 (2004).
- Li, Y. S., Haga, J. H., Chien, S. Molecular basis of the effects of shear stress on vascular endothelial cells. J. Biomech. 38, 1949-1971 (2005).
- Usami, S., Chen, H. H., Zhao, Y., Chien, S., Skalak, R. Design and construction of a linear shear stress flow chamber. Ann. Biomed. Eng. 21, 77-83 (1993).
- Helmke, B. P. Molecular control of cytoskeletal mechanics by hemodynamic forces. Physiology (Bethesda). 20, 43-53 (2005).
- Gower, R. M., Wu, H., Foster, G. A., Devaraj, S., Jialal, I., Ballantyne, C. M., Knowlton, A. A., Simon, S. I. Cd11c/cd18 expression is upregulated on blood monocytes during hypertriglyceridemia and enhances adhesion to vascular cell adhesion molecule-1. Arterioscler. Thromb. Vasc. Biol. 31, 160-166 (2011).
- Wu, H., Gower, R. M., Wang, H., Perrard, X. Y., Ma, R., Bullard, D. C., Burns, A. R., Paul, A., Smith, C. W., Simon, S. I., Ballantyne, C. M. Functional role of cd11c+ monocytes in atherogenesis associated with hypercholesterolemia. Circulation. 119, 2708-2717 (2009).
- Bussolari, S. R., Dewey, C. F., Gimbrone, M. A. Apparatus for subjecting living cells to fluid shear stress. The Review of scientific instruments. 53, 1851-1854 (1982).
