Activation of the ROCK1 Branch of the Transforming Growth Factor-β Pathway Contributes to RAGE-Dependent Acceleration of Atherosclerosis in Diabetic ApoE-Null Mice

Abstract

Rationale: The multiligand RAGE (receptor for advanced glycation end products) contributes to atherosclerosis in apolipoprotein (Apo)E-null mice. Objective: To delineate the specific mechanisms by which RAGE accelerated atherosclerosis, we performed Affymetrix gene expression arrays on aortas of nondiabetic and diabetic ApoE-null mice expressing RAGE or devoid of RAGE at nine weeks of age, as this reflected a time point at which frank atherosclerotic lesions were not yet present, but that we would be able to identify the genes likely involved in diabetes-and RAGE-dependent atherogenesis. Methods and Results: We report that there is very little overlap of the genes that are differentially expressed both in the onset of diabetes in ApoE-null mice, and in the effect of RAGE deletion in diabetic ApoE-null mice. Pathway-Express analysis revealed that the transforming growth factor-␤ pathway and focal adhesion pathways might be expected to play a significant role in both the mechanism by which diabetes facilitates the formation of atherosclerotic plaques in ApoE-null mice, and the mechanism by which deletion of RAGE ameliorates this effect. Quantitative polymerase chain reaction studies, Western blotting, and confocal microscopy in aortic tissue and in primary cultures of murine aortic smooth muscle cells supported these findings. Conclusions: Taken together, our work suggests that RAGE-dependent acceleration of atherosclerosis in ApoE-null mice is dependent, at least in part, on the action of the ROCK1 (rho-associated protein kinase 1) branch of the transforming growth factor-␤ pathway. (Circ Res. 2010;106:1040-1051.)Key Words: AGE Ⅲ atherosclerosis Ⅲ RAGE T he multiligand RAGE (receptor for advanced glycation end products) contributes to atherosclerosis in ApoE-null mice in both the nondiabetic and diabetic states. Previous studies using soluble RAGE or homozygous RAGE-null mice showed that blockade or deletion of RAGE resulted in reduction in atherosclerotic lesion area and complexity compared to control animals. [1][2][3][4][5][6] In parallel, significant downregulation of inflammatory mediators and matrix metalloproteinases was evident in ApoE-null mice aortas devoid of RAGE compared to those of RAGE-expressing ApoE-null mice.Although these findings suggested that RAGE modulated inflammatory gene expression in ApoE-null mouse aorta, they did not reveal the broader pathways by which RAGE contributed to atherosclerosis. We performed Affymetrix gene expression arrays on aortas of nondiabetic and diabetic ApoE-null mice expressing RAGE or devoid of RAGE.Aortas were harvested in the very early stages of atherogenesis, as our goal was not to identify differential genes and products expression in the atherosclerotic lesion, but to identify RAGE-dependent regulatory mechanisms in the aorta before the development of frank plaques. MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Animal StudiesMale ApoE-null mice in the C57BL/6 background were purchased from J...

RAGE biology, atherosclerosis and diabetes

“…In these mice, RAGE protein and mRNA expression is increased in the vascular endothelium [64,99]. We [64] and others [66,100] have recently shown that in diabetic RAGE − / − /apoE − / − double KO (knockout) mice, the absence of RAGE was associated with a significant attenuation of atherosclerotic plaque accumulation. The reduction in atherogenesis is associated with decreased accumulation of macrophages and T-lymphocytes, reduced aortic expression of the NFkB subunit p65 and decreased expression of inflammatory cytokines and adhesion molecules, including VCAM-1, MCP-1 and certain NADPH oxidase subunits such as gp91 phox , p47 phox and rac-1.…”

“…NF-κB up-regulates multiple cellular signalling cascades, such as NADPH oxidase, CDC42, RAC-1, the MAPKs, p21 ras , ERKs p38 MAPK and PKC [56,57,59,60]. This causes increased production of numerous growth factors and cytokines including VCAM-1, ICAM-1 (intercellular adhesion molecule-1), E-selectin, TGF-β1 (transforming growth factor-β1), CTGF (connective tissue growth factor), PDGF (plateletderived growth factor), TNF-α (tumour necrosis factor-α), IL-1 and IL-6 [61][62][63][64][65][66], all of these molecules having been implicated in the development and progression of a variety of diabetic complications.…”

Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization

“…75). In vascular cells, this interaction results in the activation of a number of intracellular pathways, including PKC/Rho (16,17,76). The prototype therapeutic agent aminoguanidine is known to prevent AGE formation by reacting with derivatives of early glycation products, such as 3-deoxyg-lucosone (reviewed in Ref.…”

“…The underlying mechanism behind this effect is in part due to activation of the PKC/Rho/Rho-kinase pathway by glucose, which results in calcium sensitization by inhibiting myosin light chain phosphatase (12)(13)(14)(15). The activation of PKC and Rho in smooth muscle by hyperglycemia is thought to be dependent on the formation of advanced glycation end products (AGEs) 2 (16,17). In addition to calcium sensitization, activation of the Rho/ Rho-kinase pathway in vascular smooth muscle cells promotes actin polymerization, an effect that plays a major role in the regulation of smooth muscle gene expression and activates myocardin-related transcription factors (MRTFs, also known as MKL1/2), co-factors to serum response factor (SRF) (18,19).…”

Overexpression of Receptor for Advanced Glycation End Products Induces Monocyte Chemoattractant Protein-1 Expression in Rat Vascular Smooth Muscle Cell Line

“…Recently, it has been reported that Src tyrosine kinase activated by RAGE is involved in a series of cellular responses in porcine aortic VSMC, including activation of MAP kinases and STAT3 as well as NF-B-dependent proinflammatory gene expression 21) . Very recently, it has been shown that RAGE activation is involved in the activation of the TGF--ROCK1 pathway in mouse aortic VSMC 22) . In contrast, several previous studies have shown that cultured VSMC barely expressing RAGE were unable to respond to RAGE ligands 5,23) .…”