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Proteomic analysis of shear stress-m...

The Medical College of Wisconsin.

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Proteomic analysis of shear stress-mediated protection from apoptosis in endothelial cells.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Proteomic analysis of shear stress-mediated protection from apoptosis in endothelial cells./
作者:	Freed, Julie K.
面頁冊數:	164 p.
附註:	Adviser: Andrew S. Greene.
Contained By:	Dissertation Abstracts International70-04B.
標題:	Biology, Animal Physiology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3354446
ISBN:	9781109126921

Proteomic analysis of shear stress-mediated protection from apoptosis in endothelial cells.
Freed, Julie K.

Proteomic analysis of shear stress-mediated protection from apoptosis in endothelial cells. - 164 p.

Adviser: Andrew S. Greene.

Thesis (Ph.D.)--The Medical College of Wisconsin, 2008.

Multiple studies have shown that the hemodynamic force of shear stress has pro-angiogenic effects within the microvasculature. Studies have also suggested that this positive angiogenic affect may be attributed to the up-regulation of survival pathways which provide protection from apoptotic stimuli such as TNF-alpha. This protection creates a favorable environment for endothelial cell survival that accommodates new vessel growth. The mechanism by which shear stress disrupts the TNF-alpha pathway remains poorly understood. Study of how these functional pathways interact within endothelial cells will aid in our overall understanding of cellular survival thus favoring capillary growth. We hypothesize that shear stress activates a series of transduction signals that ultimately interact and modify the signaling pathway of TNF-alpha. Furthermore, we hypothesize that certain membrane-associated changes account for the activation of shear stress-induced survival pathways and inhibition of these pathways will eliminate the protective effect from TNF-alpha.

ISBN: 9781109126921Subjects--Topical Terms:

1017835
Biology, Animal Physiology.

Proteomic analysis of shear stress-mediated protection from apoptosis in endothelial cells.
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100 1 $a Freed, Julie K. $3 1021877
245 1 0 $a Proteomic analysis of shear stress-mediated protection from apoptosis in endothelial cells.
300 $a 164 p.
500 $a Adviser: Andrew S. Greene.
500 $a Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 1998.
502 $a Thesis (Ph.D.)--The Medical College of Wisconsin, 2008.
520 $a Multiple studies have shown that the hemodynamic force of shear stress has pro-angiogenic effects within the microvasculature. Studies have also suggested that this positive angiogenic affect may be attributed to the up-regulation of survival pathways which provide protection from apoptotic stimuli such as TNF-alpha. This protection creates a favorable environment for endothelial cell survival that accommodates new vessel growth. The mechanism by which shear stress disrupts the TNF-alpha pathway remains poorly understood. Study of how these functional pathways interact within endothelial cells will aid in our overall understanding of cellular survival thus favoring capillary growth. We hypothesize that shear stress activates a series of transduction signals that ultimately interact and modify the signaling pathway of TNF-alpha. Furthermore, we hypothesize that certain membrane-associated changes account for the activation of shear stress-induced survival pathways and inhibition of these pathways will eliminate the protective effect from TNF-alpha.
520 $a In order to determine how shear stress modulates protection from TNF-alpha, endothelial cells were incubated with No-monomethyl- L-arginine, (LNMA) an endothelial nitric oxide synthase inhibitor prior to quantification of cleaved caspase 3, a commonly used indicator of apoptosis. Results from this study suggested that cells pre-conditioned with 18hrs of shear stress, (10dynes/cm2) while under the influence of LNMA, still protect against subsequent TNF-alpha treatment. However when cells were first incubated with cycloheximide, a protein synthesis inhibitor, shear stress-mediated protection from TNF-alpha was lost. This data revealed that the protective effect of shear stress may be due to the upregulation of anti-apoptotic proteins.
520 $a We then utilized a new strategy to isolate TNF-alpha associated proteins from sheared and non-sheared cells in order to quantitatively analyze differences in protein abundance. The goal of this new method is to preferentially label the N-terminal alpha-amino groups of intact proteins allowing the internal alpha-amino groups to remain free to react with chemical crosslinking reagents. The convergence of these methodologies allows biotinylated ligands to bind to their receptors within the cell membrane followed by removal of the crosslinked complex from cell lysate. To verify this method, biotinylated TNF-alpha was administered to endothelial cells, crosslinked, then analyzed via liquid chromatography tandem mass spectrometry (LC MS/MS) following recovery over immobilized streptavidin. Combination of the results from four separate experiments revealed a total of 100 unique proteins were associated with the TNF-alpha signaling complex. Based on information provided by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, at least 7 of the 13 known TNF-alpha apoptotic proteins were identified.
520 $a In order to quantify differences in protein abundance between sheared and nonsheared cells following treatment with biotinylated TNF-alpha, the isolated proteins underwent isotopic labeling via tryptic digestion in either H216O or H218O. LC MS/MS analysis of 1:1 mixtures of sheared versus non-sheared proteins isolated using biotinylated TNF-alpha revealed one protein, CARD9, was increased 1.6-fold in sheared cells treated with TNF-alpha. Recent studies indicated that CARD9 is involved in NF-kappaB activation and may play a role in the inhibition of apoptosis.
520 $a We then explored the possibility of a switching mechanism in which sheared cells exposed to TNF-alpha redirect signaling to proceed down the inflammatory NF-kappaB pathway as opposed to the apoptotic pathway. Western blot analysis of Tradd and Fadd, TNFR1 docking proteins that result in either NF-kappaB or apoptotic signaling respectively, showed that following shear stress endothelial cell exposure to TNF-alpha does not result in the activation of Tradd over Fadd, thus signaling is not switched near TNFR1. Further downstream analysis of NF-kappaB activation revealed that NF-kappaB is not activated following 18hrs of shear stress (10dynes/cm2) and 12hrs of TNF-alpha treatment (10ng/mL).
520 $a We also evaluated the impact of shear stress on initiating survival pathways through phospholipid activation, specifically phosphatidylserine (PS). A significant loss of shear stress protection from TNF-alpha was seen in endothelial cells grown in serine-free media through quantification of cleaved caspase 3 using Western blot. A new method was also implemented to isotopically label isolated PS from endothelial cells in order to quantify amounts of PS via electrospray ionization time-of-flight (ESI-ToF). Increases in PS measured by ESI-ToF were not observed in sheared versus non-sheared cells. The activation of the Akt pathway, a known survival pathway within endothelial cells, was shown to be activated by shear stress (10dynes/cm2, 1hr) however this activation was inhibited in sheared cells grown in serine-free media. Therefore although the measurement of PS using mass spectrometry did not suggest an increase in PS, the role of this phospholipid may still contribute to protective mechanisms within the cell.
590 $a School code: 0495.
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710 2 $a The Medical College of Wisconsin. $3 1021876
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790 1 0 $a Greene, Andrew S., $e advisor
791 $a Ph.D.
792 $a 2008
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