The impact of biomechanical forces on the differentiation of vascular cells during arteriogenesis [Elektronische Ressource] / presented by Elena Demicheva
Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the RupertoCarola University of Heidelberg, Germany
for the degree of Doctor of Natural Sciences Presented by Diplom – Biochemist Elena Demicheva born in Moscow, Russian Federation Oralexamination April, 28th, 2008
Culture of human umbilical vein endothelial cells (HUVECs)
Perfusion of isolated branches of mouse mesenteric artery
3.1.4.
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3.1.3.
Visualization of the arterial system
3.1.2.
3.1.1.
Ear artery ligation model
Hindlimb ischemia model
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In vivoin situ
3.3.3.
3.3.1.
3.3.2.
3.3.5.
3.3.4.
Transformation of competent bacteria
3.3.5.1.
3.2.5.
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TOPO cloning
Quantitative realtime PCR
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Isolation of total RNA
RTPCR
Measurement of RNA/cDNA concentration
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Stimulation of cultured cells with Idebenone or glucose oxidase
Culture of mouse smooth muscle cells
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Plating and passage of smooth muscle cells (enzymatic hydrolysis)
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3.2.2.
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Application of shear stress and cyclic stretch
3.3.6.2.
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3.4.1.
3.4.2.
3.4.3.
3.4.4.
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4.1.1.
4.1.2.
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4.2.1.
4.2.2.
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4.3.1.
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4.4.1.
Administration of the decoy oligodeoxynucleotides
Immunohistological analysis
Dihydroethidium staining
lectin staining
ELISA
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/ Arteriogenic remodelling is characterized by an increase in diameter of collateral arterioles after femoral artery ligation MCP1 is one of the first molecules upregulated during the onset of arteriogenesis 0 Arteriogenic remodelling in the mouse ear is charac terized by an increase in number and diameter of corkscrewlike arterioles after artery occlusion Adaptive remodelling of collateral arterioles after ear artery ligation is accompanied by an increased expression of MCP1 and ICAM1 Proarteriogenic perfusion conditions upregulate MCP1 expression in vascular smooth muscle cells
0 in vitroCyclic stretch rather than shear stress upregulates MCP1 expression in cultured vascular cells
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4.5.1.
4.5.2.
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4.6.1.
4.6.2.
4.6.3.
4.6.4.
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1 "1 ( Expression of MCP1 induced by proarteriogenic perfusion conditions is dependent on the activation of AP1 Activation of AP1 is critical for straininduced MCP1 expression .2- ( Cyclic stretch increases production of ROS in cultured smooth muscle cells Formation of ROS is necessary for MCP1 expression induced by proarteriogenic flow conditions An increased ROS formation is associated with collateral arterioles undergoing arteriogenesis Expression of MCP1 can be induced by an increase i n exogenous ROS formation 1 "1 ( -1