Protocatechuic aldehyde inhibits migration and proliferation of vascular smooth muscle cells and intravascular thrombosis

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Abstract

The migration and proliferation of vascular smooth muscle cells (VSMCs) and formation of intravascular thrombosis play crucial roles in the development of atherosclerotic lesions. This study examined the effects of protocatechuic aldehyde (PCA), a compound isolated from the aqueous extract of the root of Salvia miltiorrhiza, an herb used in traditional Chinese medicine to treat a variety of vascular diseases, on the migration and proliferation of VSMCs and platelets due to platelet-derived growth factor (PDGF). DNA 5-bromo-2′-deoxy-uridine (BrdU) incorporation and wound-healing assays indicated that PCA significantly attenuated PDGF-induced proliferation and migration of VSMCs at a pharmacologically relevant concentration (100 μM). On a molecular level, we observed down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathways, both of which regulate key enzymes associated with migration and proliferation. We also found that PCA induced S-phase arrest of the VSMC cell cycle and suppressed cyclin D2 expression. In addition, PCA inhibited PDGF-BB-stimulated reactive oxygen species production in VSMCs, indicating that PCA’s antioxidant properties may contribute to its suppression of PDGF-induced migration and proliferation in VSMCs. Finally, PCA exhibited an anti-thrombotic effect related to its inhibition of platelet aggregation, confirmed with an aggregometer. Together, these findings suggest a potential therapeutic role of PCA in the treatment of atherosclerosis and angioplasty-induced vascular restenosis.

Highlights

► Protocatechuic aldehyde (PCA) inhibits ROS production in VSMCs. ► PCA inhibits proliferation and migration in PDGF-induced VSMCs. ► PCA has anti-platelet effects in ex vivo rat whole blood. ► We report the potential therapeutic role of PCA in atherosclerosis.

Introduction

A variety of natural products with antioxidant properties have been used as traditional remedies for atherosclerosis. The herb Salvia miltiorrhiza Bunge (Labiatae) has shown beneficial effects in coronary heart disease, cerebrovascular disease, liver cirrhosis, and insomnia [1]. Although the mechanisms by which S. miltiorrhiza may improve atherosclerosis remain unclear, both the extract itself and isolated compounds of S. miltiorrhiza have demonstrated multiple antioxidant mechanisms [2], [3], [4], [5].

The abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) in arterial walls are important factors in the development of atherosclerosis as well as restenosis after angioplasty [2]. Although these processes are triggered by multiple cytokines and growth factors, one of the principal regulators of chemoattraction and mitogenesis of VSMCs is platelet-derived growth factor (PDGF)-BB, the expression of which is increased in atherosclerotic lesions [3], [4], [5]. The PDGF-induced mitogenesis signaling pathway has already been well characterized. For example, phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) are the two major PDGF signaling pathways and are linked to numerous cellular process, including proliferation and migration [2]. ERK 1/2 and PI3K pathways have also been shown to be important for PDGF-BB-induced cell cycle progression in VSMCs.

Several studies have reported that reactive oxygen species (ROS) generation occurs during restenosis after angioplasty [6], [7], [8] and that antioxidants attenuate neo-intimal hyperplasia [7], [9], [10], [11]. Furthermore, recent evidence indicates that PDGF itself stimulates ROS production in VSMCs [6], [12]. Though ROS were previously thought to be destructive to cells, numerous studies have shown them to be crucial messengers in the transduction of VSMCs’ responses to PDGF, especially in terms of activating MAPK and Akt pathways. However, ROS can induce mitochondrial DNA damage, which has been reported to be associated with atherosclerosis [13]. These findings suggest that ROS may play a central role in VSMC proliferation and migration, and indicate that antioxidant substances may have beneficial effects in atherosclerosis.

Platelets also play an important role in thrombus formation at the site of damaged blood vessels, resulting in many cases of cardiovascular and cerebrovascular diseases [14]. Collaborative meta-analysis of randomized trials has shown that anti-platelet therapy prevents serious vascular events in a wide range of patients with atherosclerosis.

Protocatechuic aldehyde (PCA), a water-soluble compound isolated from the root of the herb S. miltiorrhiza, has been reported to protect against TNF-α-induced endothelial cell injury by suppressing ICAM-1 and VCAM-1 expression in human umbilical vein endothelial cells [15], as well as stimulating free radical scavenging activity in RAW264.7 macrophages [16]. To the best of our knowledge, however, no studies have been published that examine the influence of PCA on VSMC migration and proliferation.

In this study, we investigated the effects of PCA’s antioxidant properties on PDGF-induced VSMC proliferation and migration. Furthermore, we examined PCA’s influence on PI3K/Akt and ERK 1/2 pathways, which regulate cellular proliferation and migration. Finally, we evaluated the anti-thrombotic effect of PCA through inhibition of ADP-induced platelet aggregation.

Section snippets

Ethics statement

This study was conducted in accordance with the Institutional Animal Care and Use Committee of Yonsei University Health System based on the Laboratory Animal Manual and the “Guide for the care and use of laboratory animals” edited by the National Research Council of the National Academies (permit number 2010-0187). All animal studies were performed in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care.

Reagents

We purchased recombinant rat PDGF-BB from R&D

PCA inhibition of ROS production and PDGF-BB-induced VSMC proliferation and migration

Recent evidence supports the hypothesis that PDGF increases intracellular ROS levels in VSMCs and that both PDGF-induced VSMC proliferation and migration are ROS-dependent. As shown in Fig. 1A, 20 ng/mL PDGF-BB significantly increased ROS (75.06%) compared to control cells (42.06%). Furthermore, PCA considerably neutralized the PDGF-BB-induced ROS accumulated in VSMCs (from 75.06% to 4.07%). Quantitative analysis of VSMC proliferation was measured by BrdU incorporation (Fig. 1B). When VSMCs were

Discussion

PCA is a water-soluble antioxidant phenolic aldehyde isolated from the root of S. miltiorrhiza. In traditional Chinese medicine, this herb has been used to treat vascular diseases for centuries with no serious adverse effects reported in the scientific literature [1]. The herb’s therapeutic utility in cardiovascular disease has been attributed to improved microcirculation, vasodilation, anti-coagulation, and anti-inflammation [21], [22]. Recently, several chemical compounds have been isolated

Acknowledgments

This work was supported by the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs (Grant numbers: A085136 [to E.J.L]).

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