Mixed-Lineage Kinase 3 Deficiency Promotes Neointima Formation Through Increased Activation of the RhoA Pathway in Vascular Smooth Muscle Cells

Abstract

Objective Mitogen Activated Protein Kinase (MAPK) pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a MAP kinase kinase kinase (MAP3K) that activates multiple MAPK pathways, and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia, and to elucidate the underlying mechanisms. Approach and Results Neointima formation was analyzed after endothelial denudation of carotid arteries from wild-type (WT) and Mlk3−/− mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from Mlk3−/− mice compared to WT mice. Furthermore, MLK3 deficiency increased activation of p63RhoGEF, RhoA and Rho-kinase (ROCK) in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated ROCK activation. Moreover, JNK activation was decreased in MLK3 deficient VSMC and proliferation of WT but not MLK3 KO cells treated with a JNK inhibitor was attenuated. Conclusions We demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation, by binding to and inhibiting activation of p63RhoGEF, a RhoA activator. In MLK3 deficient cells, activation of p63RhoGEF proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63RhoGEF interaction, which is attenuated by feedback from activated JNK.

Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

“…As the main signaling of canonical TGF-b pathway, Smad2 pathway has been demonstrated to be involved in the process of vascular remodeling [46,47]. And as a major noncanonical signaling, ERK pathway regulates cell proliferation, apoptosis, migration and MMP expression, all of these are significantly implicated in the pathogenesis of vascular remodeling [4,34]. Furthermore, complete inhibition of ERK pathway alone is able to totally downregulate MMP9 expression [34].…”

“…Inflammatory cells, such as macrophages, play a critical role in vascular remodeling of carotid artery in response to flow changes [3,4,37,38]. Inflammatory cells, known for their secretion of cytokines and proteinases, including MMP, are emerging as key modulators for various tissue remodeling [4,34].…”

“…Macrophage infiltration is an important event for vascular remodeling [3,4,37,38]. Therefore, we examined the role of TGF-b in SMC for macrophage infiltration during vascular remodeling.…”

“…MMPs have been prominently involved in the process of vascular remodeling [4,34]. Furthermore, the importance of MMP in the carotid remodeling is illustrated by the findings that inhibition of MMP can prevent the remodeling process [40].…”

“…Media thickening and macrophage infiltration are the major structural events that may lead to lumen narrowing in many clinical cardiovascular diseases [3,4]. The changes of wall mass and geometry both require macrophage infiltration, SMC proliferation, and extracellular matrix (ECM) deposition [5,6].…”