There is increasing support for the idea that inflammation plays a major role in the development of atherosclerosis. Peroxisome proliferator-activated receptors (PPARgamma) are ligand-activated transcription factors that form a subfamily of the nuclear gene family. PPARgamma activators inhibit adhesion molecules expression in activated endothelial cells and significantly reduce monocyte/macrophage homing to atherosclerotic plaques. We found that steady laminar flow activates ERK5, and increases PPARgamma transcriptional activation. In addition, to utilize the newly identified Hinge-helixl region of PPARgamma fragment (Hin-H1 fragment), which specifically disrupts ERK5/PPARgamma interaction and inhibits activated ERK5-mediated PPARgamma activation, will provide a great tool to explore the specific role of ERK5 and PPARgamma interaction in laminar flow-mediated anti-inflammatory effect. Based on our exciting preliminary data that ERK5 activation increases PPARgamma activity and that this may inhibit the expression of adhesion molecules, we hypothesize that laminar flow acts as an anti-inflammatory modulator by increasing PPARgamma transcriptional activity, and decreasing VCAM- 1 expression in endothelial cell. To prove the contribution of PPARgamma transcriptional activity (which is regulated by ERK5) to the atheroprotective effect of flow in endothelial cells we propose three aims.
Aim 1. Using cultured endothelial ceils, define the role of ERK5/PPARgamma on TNFalpha induced NF-kappaB, VCAM-1 promoter activation and subsequent VCAM-1 expression.
Aim 2 : Using an ex vivo organ culture system of intact aorta, determine the role of PPARgamma and ERK5 in inflammation-related expression of adhesion molecules Aim 3: Using an atherosclerosis model, determine endothelial ERK5, JNK, NF-kappaB activation and VCAM- 1 expression in vivo by using en face confocal microscopy, and detect the role of endothelial ERK5 in atherosclerosis formation using gain and loss of function mice.
In aim 1 we will show the importance of ERK5/PPARgamma interaction to regulate laminar flow-mediated anti-inflammatory effect using Hin-H 1 fragment and ERK5 siRNA in adenovirus vector.
In aim 2 we will utilize the organ culture system, and determine the role of ERK5-PPARgamma/ in flow in intact vessels.
In aim 3 we will use endothelial specific constitutively active(CA)-MEK5 or ERK5 knock out in LDLR-/- knock out(KO) mice, which has been characterized by atherosclerosis formation. We anticipate that CA-MEK5 will inhibit and ERK5 KO will promote atherosclerosis formation. These experiments will provide a new molecular mechanism for the anti-inflammatory effect of PPARgamma and flow associated with ERK5 activity.
