Low-Density Lipoproteins Induce Heat Shock Protein 27 Dephosphorylation, Oligomerization, and Subcellular Relocalization in Human Vascular Smooth Muscle Cells

Abstract

Objective-High levels of circulating low-density lipoproteins (LDL) are a major atherosclerotic risk factor. The effects of intimal LDL on vascular smooth muscle cell (VSMC) phenotype and function during plaque remodeling and vascular repair are not fully understood. We have investigated whether exposure of VSMC to LDL induces changes on the proteomic profile of the heat shock protein (HSP) family-molecular chaperones involved in atherosclerosis. Methods & Results-2D electrophoresis demonstrates that LDL modifies the proteomic profile of HSP27 (HSPB1).Western blot analysis evidenced a significant HSP27 dephosphorylation after exposure of cells to native LDL (nLDL) and aggregated-LDL (agLDL) for 24 hours (PϽ0.05). Dephosphorylation of HSP27 was not related to changes in p38 MAPK phosphorylation. Both nLDL and agLDL induced relocalization of unphosphorylated HSP27 to the tip of actin stress fibers and focal adhesion structures in VSMC. During cell adhesion, phospho-HSP27 was located at the cell surface contact region in LDL-treated cells, whereas it remained cytosolic in control cells. Immunohistochemistry studies showed that phosphorylated HSP27 is rarely found in lipid-rich areas of atherosclerotic plaques in human coronary arteries. (oxidation, glycosylation, aggregation). Modified LDL have been associated with changes in endothelial function, 2 as well as with migration, proliferation, and apoptosis of vascular smooth muscle cells (VSMCs). 3 Migration and proliferation of VSMCs, as well as extracellular matrix production and catabolism, are important events in the development of atherosclerosis and arterial remodeling. 4 VSMCs acquire or lose various functions to be able to fulfill the requirements for intimal remodeling. 5 Previous studies from our group demonstrated that LDL induce changes on VSMC gene expression and phenotype, leading to alterations in vascular function. 6 -8 We have recently demonstrated that aggregated LDL (agLDL) impair VSMC migration and wound repair after injury. 9 In response to stress, cells activate cytoprotective pathways. Heat shock proteins (HSP) are molecular chaperones that have the ability to protect proteins from damage induced by factors such as free radicals, heat, or ischemia. It has been demonstrated that HSP are highly expressed in atherosclerotic lesions of humans, rabbits, and apolipoprotein E-deficient mice. 10 Interestingly, HSP90 (HSP90AA1) expression is induced in VSMC by oxidative stress 11 and plays a role in the maintenance of endothelial barrier integrity by binding endothelial nitric oxide synthase and guanylyl cyclase (reviewed in Antonova et al 12 ). Overexpression of HSP70 (HSPA1A) in mouse heart increases resistance to ischemic injury. 13 Small HSP can also protect against ischemic injury in cardiomyocytes. 14 Among small HSP, HSP27 (HSPB1) acts as a molecular chaperone in vitro 15 and is also involved in F-actin assembly. 16,17 In smooth muscle, HSP27 is constitutively expressed and, when phosphorylated, colocalizes with contractile proteins. 18 In th...

The Use of High-Throughput Technologies to Investigate Vascular Inflammation and Atherosclerosis

“…Nevertheless, other studies, addressing more specific questions, did investigate signaling pathways by proteomic approaches: 2-DE identified the correlation of the strongly anti-apoptotic heat shock protein (HSP)-27 phosphorylation level with cardiovascular diseases, by identifying LDL as an important regulator of HSP27 phosphorylation involving p38 -mitogen-activated protein kinase signaling in VSMCs. 92 Moreover, 2-DE and MS revealed that paracrine secretion of platelet-derived growth factor-BB by ECs is implicated in the pathophysiological vascular remodeling induced by low shear stress, involving the phosphorylation of extracellular signalregulated kinases (ERK)1 and 2, which belong to the mitogen-activated protein kinase family. 93 In addition, the importance of ERK1 and ERK2 phosphorylation was shown in vitro in bovine aortic ECs after stimulation with oxidized phospholipids using liquid chromatography-mass spectrometry analysis.…”

Proteomic Signature of Thrombin-Activated Platelets After In Vivo Nitric Oxide–Donor Treatment

“…Before protein separation by 2-dimensional gel electrophoresis (2-DE), sample contaminants were removed, and protein concentration in the extracts was measured. 18 Protein extracts of control platelets and thrombin-stimulated platelets obtained from animals treated without or with NOd were separated by 2-DE. 18,19 Isoelectric focusing was performed using the Protean-IEF cell (Bio-Rad).…”

“…18 Protein extracts of control platelets and thrombin-stimulated platelets obtained from animals treated without or with NOd were separated by 2-DE. 18,19 Isoelectric focusing was performed using the Protean-IEF cell (Bio-Rad). Protein loads were applied on linear immobilized pH gradient strips and separated by 2-DE.…”

“…18 A Tris-soluble (cytosolic) fraction and a ureadetergent soluble (cytoskeleton/membrane) fraction were obtained. Before protein separation by 2-dimensional gel electrophoresis (2-DE), sample contaminants were removed, and protein concentration in the extracts was measured.…”

HSP27 is Commonly Expressed in Cervical Intraepithelial Lesions of Brazilian Women

“…This protein could increase the antioxidant defense of cells by increasing glutathione cell content neutralizing the toxic effects of oxidized proteins by its chaperon activity (Uçar et al, 2012). The yours cytoprotective effects has also been attributed to its capacity to bind and stabilize actin polymerization (García-Arguinzonis et al, 2010). The localization of Hsp27 been demonstrated in bovine endothelial cells, in which it probably contributes to the regulation of membrane associated F-actin polymerization (Lavoie et al, 1995).…”