Molecular Mechanisms of High Glucose-Induced Vascular Endothelial Growth Factor Expression in Retinal Endothelial Cells

Hdl Handle:
http://hdl.handle.net/10675.2/556468
Title:
Molecular Mechanisms of High Glucose-Induced Vascular Endothelial Growth Factor Expression in Retinal Endothelial Cells
Authors:
Platt, Daniel H.
Abstract:
Studies in diabetic patients, experimental animal models and tissue culture cells treated with high glucose have shown a close association between pathologic vascular growth, over-expression o f the angiogenic factor vascular endothelial growth factor .5 (VEGF) and oxidative stress. Studies o f diabetic patients and high glucose treated cells have also shown increased levels o f tyrosine nitration, a marker for the formation o f the reactive nitrogen species peroxynitrite. Excess formation o f reactive oxygen/nitrogen species has been shown to activate two transcription factors that regulate the expression o f VEGF, hypoxia-inducible factor-1 (HIF-1) and signal transducer and activator o f transcription 3 (STAT3). These observations suggest that diabetes causes increases in VEGF expression due to the effects o f high glucose in stimulating the formation o f peroxynitrite, which leads to the activation o f the transcription factors HIF-1 and/or STAT3 and increases in VEGF expression. This hypothesis was tested by experiments using primary cultures o f retinal endothelial cells treated with peroxynitrite or high glucose. Both treatments increased VEGF mRNA and protein levels. Further, pretreatment with the specific peroxynitrite decomposition catalyst FeTPPs blocked the increase in VEGF expression. To determine if HIF-1 and/or STAT3 play a role in the peroxynitrite-induced VEGF expression, studies were done to analyze the activation patterns o f both transcription factors. These studies showed that peroxynitrite had no effect on the activation or nuclear translocation o f HIF-1 a , but did induce a rapid activation and nuclear translocation o f STAT3. To further explore the role o f STAT3 in the VEGF expression, cells were treated with peroxynitrite or high glucose in the presence or absence o f an adenoviral vector expressing dominant-negative STAT3. Overexpression o f the dominant-negative STAT3 blocked the effects o f either peroxynitrite or high glucose in increasing VEGF mRNA. Further, treatment with FeTPPS blocked the effects o f high glucose in stimulating activation o f STAT3. A non-receptor tyrosine kinase, cSrc, has been shown to play a role in the activation o f STAT3 as well as the induction o f VEGF expression during tumor angiogenesis. To determine if cSrc plays a role in STAT3 regulated VEGF transcriptional activation, retinal endothelial cells were transduced with an adenovirus over-expressing a constitutively active Src (vSrc). The vSrc transduction induced activation o f STAT3 and increased VEGF expression. Further, FeTPPs blocked the effects o f peroxynitrite and high glucose in stimulating activation o f cSrc. Additionally, the Src inhibitor PP1 blocked the effects o f peroxynitrite and high glucose in increasing VEGF mRNA and protein expression. This work is the first to show that 1) high glucose-induced peroxynitrite formation increases VEGF expression, 2) STAT3 activation by high glucose-induced peroxynitrite formation regulates VEGF expression and 3) cSrc activation by high glucose-induced peroxynitrite formation activates STAT3 and increases VEGF expression.
Affiliation:
Vascular Biology Center
Issue Date:
Oct-2004
URI:
http://hdl.handle.net/10675.2/556468
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/305097764?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorPlatt, Daniel H.en
dc.date.accessioned2015-06-05T18:56:17Zen
dc.date.available2015-06-05T18:56:17Zen
dc.date.issued2004-10en
dc.identifier.urihttp://hdl.handle.net/10675.2/556468en
dc.description.abstractStudies in diabetic patients, experimental animal models and tissue culture cells treated with high glucose have shown a close association between pathologic vascular growth, over-expression o f the angiogenic factor vascular endothelial growth factor .5 (VEGF) and oxidative stress. Studies o f diabetic patients and high glucose treated cells have also shown increased levels o f tyrosine nitration, a marker for the formation o f the reactive nitrogen species peroxynitrite. Excess formation o f reactive oxygen/nitrogen species has been shown to activate two transcription factors that regulate the expression o f VEGF, hypoxia-inducible factor-1 (HIF-1) and signal transducer and activator o f transcription 3 (STAT3). These observations suggest that diabetes causes increases in VEGF expression due to the effects o f high glucose in stimulating the formation o f peroxynitrite, which leads to the activation o f the transcription factors HIF-1 and/or STAT3 and increases in VEGF expression. This hypothesis was tested by experiments using primary cultures o f retinal endothelial cells treated with peroxynitrite or high glucose. Both treatments increased VEGF mRNA and protein levels. Further, pretreatment with the specific peroxynitrite decomposition catalyst FeTPPs blocked the increase in VEGF expression. To determine if HIF-1 and/or STAT3 play a role in the peroxynitrite-induced VEGF expression, studies were done to analyze the activation patterns o f both transcription factors. These studies showed that peroxynitrite had no effect on the activation or nuclear translocation o f HIF-1 a , but did induce a rapid activation and nuclear translocation o f STAT3. To further explore the role o f STAT3 in the VEGF expression, cells were treated with peroxynitrite or high glucose in the presence or absence o f an adenoviral vector expressing dominant-negative STAT3. Overexpression o f the dominant-negative STAT3 blocked the effects o f either peroxynitrite or high glucose in increasing VEGF mRNA. Further, treatment with FeTPPS blocked the effects o f high glucose in stimulating activation o f STAT3. A non-receptor tyrosine kinase, cSrc, has been shown to play a role in the activation o f STAT3 as well as the induction o f VEGF expression during tumor angiogenesis. To determine if cSrc plays a role in STAT3 regulated VEGF transcriptional activation, retinal endothelial cells were transduced with an adenovirus over-expressing a constitutively active Src (vSrc). The vSrc transduction induced activation o f STAT3 and increased VEGF expression. Further, FeTPPs blocked the effects o f peroxynitrite and high glucose in stimulating activation o f cSrc. Additionally, the Src inhibitor PP1 blocked the effects o f peroxynitrite and high glucose in increasing VEGF mRNA and protein expression. This work is the first to show that 1) high glucose-induced peroxynitrite formation increases VEGF expression, 2) STAT3 activation by high glucose-induced peroxynitrite formation regulates VEGF expression and 3) cSrc activation by high glucose-induced peroxynitrite formation activates STAT3 and increases VEGF expression.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/305097764?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectVascular Endothelial Growth Factoren
dc.subjectTyrosineen
dc.subjectAdenoviridaeen
dc.subjectDiabetes Mellitusen
dc.subjectGlucoseen
dc.subjectHypoxia-Inducible Factor 1en
dc.subjectNeoplasmsen
dc.subjectNitrogenen
dc.titleMolecular Mechanisms of High Glucose-Induced Vascular Endothelial Growth Factor Expression in Retinal Endothelial Cellsen
dc.typeDissertationen
dc.contributor.departmentVascular Biology Centeren
dc.description.advisorCaldwell, Ruth B.en
dc.description.committeeMarrero, Mario B.; Lewis, Deborah L.; Pollock, Jennifer S.en
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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