The broad long term objective/goal of the proposed work is to gain insight into the mechanical and molecular mechanisms by which vascular endothelial cells (ECs) sense hemodynamic stimuli and transduce the resulting signals to post-translational modifications and transcriptional regulation.
Three specific aims are proposed to test the hypothesis that AMP-activated protein kinase (AMPK) in ECs is activated by atheroprotective flow, which in turn upregulates genes beneficial to vascular tone.
Specific Aim 1 : to delineate the biomechanical basis of atheroprotective flow in activating the AMPK kinase (AMPK)-AMPK cascade in ECs;
Specific Aim 2 : to elucidate the molecular basis underlying the transcriptional activation of atheroprotective genes by AMPK in response to atheroprotective flow;
and Specific Aim 3 : to investigate the ro|e of flow activated AMPK in vascular tone ex vivo and atheroprotection in vivo. We will use flow channels to subject a monolayer of cultured ECs to flow with variations in hemodynamic factors (magnitude, temporal/spatial gradients, frequency and amplitude) and assess the activation of AMPKK, namely camodulin kinase kinase and LKB1, and AMPK (by kinase activity assay and Western blot) as biological readouts. We will then determine the molecular basis underlying the differential gene expression (e.g. eNOS, ASS, ACC, CNP) in ECs responding to different flows. We will utilize chromatin immunoprecipitation (ChIP) assays to study the epigenetic modification of the AMPK target genes. Parallel inhibition experiments utilizing siRNA, dominant negative mutant of AMPK, and pharmalogical inhibitors will be conducted to provide supporting evidence. We will investigate gene expression pattern, NO bioavailability, and EC- dependent vascular tone as functional consequences. The use of selected breeding of gene specific (i.e., ampk-/-, apoE-/- mice) mutant mice will allow us to assess distribution and extent of atherosclerotic lesions are enhanced because of the loss-of-function of AMPK. This project will help to define the parameters and thresholds distinguishing atheroprotective from atheroprone flow patterns. Study of the molecular mechanisms by which the defined flow patterns affect vasodilation and endothelial function will contribute to defining the mechanical and chemical factors involved in causation/prevention/diagnosis/treatment of endothelial dysfunction (e.g. atherosclerosis, hypertension, diabetes).