High glucose induces plasminogen activator inhibitor-1 expression through Rho/Rho-kinase-mediated NF-κB activation in bovine aortic endothelial cells

doi:10.1016/j.atherosclerosis.2006.12.025

Atherosclerosis

Volume 196, Issue 1, January 2008, Pages 22-28

https://doi.org/10.1016/j.atherosclerosis.2006.12.025 Get rights and content

Abstract

Recently, it has become evident that elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with myocardial infarction and stroke, especially in patients with diabetes. The molecular mechanisms involved in hyperglycemia-induced PAI-1 expression in bovine aortic endothelial cells (BAEC) were investigated.

PAI-1 expression in BAEC was significantly increased in accordance with the concentration of glucose in media from 5.7 mM to 23 mM. Stimulation with high glucose (23 mM) significantly increased small GTPase RhoA activation. Pretreatment with a Rho-kinase inhibitor, Y-27632 (1–10 μM), significantly blocked high glucose-induced PAI-1 expression. NF-κB activity determined using the luciferase reporter gene assay was significantly enhanced by high glucose, and pretreatment with Y-27632 inhibited high glucose-induced PAI-1 expression at the basal level. An inhibitor of NF-κB action, namely parthenolide (0.1 μM), BAY 11-7082 (5 μM) and SN50 (1 μM), significantly blocked high glucose-mediated PAI-1 expression to a level with low glucose (5.7 mM). These data suggested that high glucose-induced PAI-1 expression in endothelial cells is mediated by NF-κB activation through the Rho/Rho-kinase pathway. Inhibition of Rho/Rho-kinase signaling might be a novel target for diabetes and metabolic syndrome.

Introduction

Plasminogen activator inhibitor-1 (PAI-1) is the major physiologic inhibitor of tissue-type plasminogen activator in plasma, and is elevated in a variety of clinical situations associated with an increased risk of atherothrombotic disorder [1]. Recent evidence indicated that increased PAI-1 production might be of particular importance in the development of vascular disease in diabetes [2] and might be a link between insulin resistance/metabolic syndrome and atherothrombosis [3].

The nuclear factor (NF)-κB family has an important function in the regulation of many genes involved in the inflammatory and proliferative responses of cells, and recent studies indicate that NF-κB is involved in the pathogenesis of atherosclerosis [4]. Several lines of evidence indicated that high glucose-induced NF-κB activation in endothelial cells (EC) [5], [6], mesangial cells [7], vascular smooth muscle cells [8] and monocytic cells [9]. Therefore, NF-κB activation is an event in response to elevated glucose, which may elicit multiple pathways contributing to the origin of hyperglycemia- or diabetes-induced EC injury; however, the signal pathway of NF-κB mediating the vascular complications of hyperglycemia is unclear.

Rho/Rho-kinase signaling was identified to occur in cardiovascular diseases including vascular remodeling and atherosclerosis [10]. We previously identified that Rho/Rho-kinase-mediated NF-κB activation was involved in CRP-induced PAI-1 expression in EC [11]. Recently, Rikitake et al. reported that hyperglycemia stimulates Rho-kinase activity via PKC- and oxidative stress-dependent pathways, leading to increased PAI-1 gene transcription [12]; however, the mechanism of downstream signaling by Rho-kinase-mediated PAI-1 expression under high glucose remains unknown.

The aim of this investigation was to clarify whether NF-κB and Rho/Rho-kinase pathways are involved in PAI-1 expression by high glucose stimulation in bovine aortic endothelial cells (BAEC). We demonstrated in this study that high glucose activates RhoA, which then activates NF-κB signaling through Rho-kinse, resulting in PAI-1 expression. These findings provide evidence for the possible involvement of Rho/Rho-kinase signaling in NF-κB activation in hyperglycemia-induced atherothrombogenesis.

Section snippets

Materials

The chemicals used were as follows: Y-27632 (Calbiochem, CA, USA), a Rho-kinase inhibitor; three kinds of inhibitors of NF-κB action, parthenolide (Calbiochem) [13], BAY 11-7082 (BIOMOL, PA, USA) [14], and SN50 (BIOMOL) [15]. All other chemicals were of the highest grade commercially available.

Cell culture

BAEC were harvested from thoracic aortas and maintained as described previously [16]. Cells were grown to confluence in DMEM (Sigma, MO, USA) with 10% fetal bovine serum (Biowest, Ringmer, UK), 100

PAI-1 expression induced by high glucose in BAEC

As shown in Fig. 1A, a significant increase in PAI-1 expression was detected after 6 h, the expression peaked at 12 h, and then declined. The levels of PAI-1 expression were dependent on glucose concentration (5.7–23 mM) (Fig. 1B); however, as shown in Fig. 1C, similar increased concentrations of mannitol did not affect PAI-1 expression.

Activation of RhoA in BAEC stimulated by high glucose

The level of the active GTP-bound form of RhoA was examined by a pull-down assay with the Rho-binding fragment of rhotekin. As shown in Fig. 2A and B, stimulation

Discussion

Inflammation is involved in all stages of atherogenesis, in which various mediators, including cytokines, chemokines, growth factors and adhesion molecules, play important roles [18]. In addition to being an independent cardiovascular disease risk factor, recent accumulating evidence implicates hyperglycemia such as postprandial hyperglycemia as a mediator of the proatherogenic effect, which leads to endothelial dysfunction and plaque instability [19]. In EC, hyperglycemia elicited

Acknowledgements

This work was supported in part by grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science. We thank Dr. T. Nakakuki for helpful discussions.

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High glucose induces plasminogen activator inhibitor-1 expression through Rho/Rho-kinase-mediated NF-κB activation in bovine aortic endothelial cells

Abstract

Introduction

Section snippets

Materials

Cell culture

PAI-1 expression induced by high glucose in BAEC

Activation of RhoA in BAEC stimulated by high glucose

Discussion

Acknowledgements

Atherosclerosis

Atherosclerosis

J Biol Chem

J Biol Chem

J Biol Chem

J Am Coll Cardiol

J Thromb Haemost

J Biol Chem

Kidney Int

Cell

Mol Cell

Gastroenterology

J Biol Chem

Plasminogen activator inhibitor-type 1: its plasma determinants and relation with cardiovascular risk

Thromb Haemost

Increased plasminogen activator inhibitor type 1 in coronary artery atherectomy specimens from type 2 diabetic compared with nondiabetic patients: a potential factor predisposing to thrombosis and its persistence

Circulation

Metabolic syndrome: a comprehensive perspective based on interactions between obesity, diabetes, and inflammation

Circulation