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Trevor Cardinal
Ph.D. Candidate
Physiological Sciences GIDP
Experimental Biology
San Francisco, California
April 1- 5, 2006
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“Targeted Disruption of Fgf2 Impairs Blood Flow Control in the Posterior Gracilis During Revascularization”
ABSTRACT
Introduction
Following induction of chronic ischemia through unilateral ligation of the femoral-saphenous artery & vein, the mouse hindlimb undergoes revascularization to re-establish perfusion to the affected area. Revascularization includes the development of new large arteries, termed collateralization, as well as the development of new capillaries and arterioles, termed angiogenesis and arteriolarization respectively. Because fibroblast growth factor 2 (Fgf2), which has a potent proliferative effect on vascular cells, is upregulated during the first week following ligation, it was thought that Fgf2 plays an important role in revascularization.
However, using Fgf2 null mice we found that Fgf2 is not necessary for vascular growth during revascularization, as determined by microangiographic assessment of collateralization and histological assessment of arteriolarization and angiogenesis. Instead, we found that Fgf2 is necessary for maintaining the capacity for blood flow control during revascularization; reactive hyperemia, an increase in blood flow following a 10-minute iliac artery occlusion, was absent at day 14 following ligation.
Methods
To evaluate the role of Fgf2 in maintaining the capacity for blood flow control in specific vascular beds, I developed a novel fluorescent microsphere-based approach to measure blood flow in individual skeletal muscles, before and after electrical stimulation. This method is involves the intra-arterial injection of 15µm fluorescent spheres; the number of fluorescent spheres that lodge in the peripheral tissues is directly proportional to the amount of blood flowing through that tissue. Absolute blood flow can be calculated by determining the arterial concentration of the spheres during the injection by sampling arterial blood distal to the injection site.
To examine the molecular mechanism underlying the impairment of blood flow control in the revascularizing hindlimb of Fgf2 null mice, I analyzed the expression of genes involved in blood flow control. RNA was isolated from the posterior gracilis muscle of wild type and Fgf2 null mice and converted to cDNA by reverse transcription. cDNA levels were determined through real-time quantitative polymerase chain reaction, in which cDNA is amplified and the fluorescence, which is emitted by fluorescent dye that intercalates within the cDNA, is used to quantify relative cDNA number.
Results
Preliminary experiments with Fgf2 null mice demonstrate that functional hyperemia, increase in blood flow following muscle stimulation, is absent in the posterior gracilis, but not in the anterior gracilis at day 14 following ligation. Additionally, preliminary gene expression analysis of the posterior gracilis muscle at day 14 following ligation demonstrates that the vasodilating ADP receptor (P2Y1) is down-regulated by nearly four fold as compared to wild type mice.
Conclusions
Because vasodilation is necessary for increases in blood flow, these data suggest that the impairment in blood flow control in the revascularizing hindlimb of the Fgf2 null mouse is due to the down-regulation of the ADP receptor.
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