Oxidative stress generated by polycyclic aromatic hydrocarbons from ambient particulate matter enhance vascular smooth muscle cell migration through MMP upregulation and actin reorganization.

BACKGROUND: Epidemiological studies have suggested that elevated concentrations of particulate matter (PM) are strongly associated with the incidence of atherosclerosis, however, the underlying cellular and molecular mechanisms of atherosclerosis by PM exposure and the components that are mainly responsible for this adverse effect remain to be established. In this investigation, we evaluated the effects of ambient PM on vascular smooth muscle cell (VSMC) behavior. Furthermore, the effects of polycyclic aromatic hydrocarbons (PAHs), major components of PM, on VSMC migration and the underlying mechanisms were examined. RESULTS: VSMC migration was significantly increased by treatment with organic matters extracted from ambient PM. The total amount of PAHs contained in WPM was higher than that in SPM, leading to higher ROS generation and VSMC migration. The increased migration was successfully inhibited by treatment with the anti-oxidant, N-acetyl-cysteine (NAC). The levels of matrix metalloproteinase (MMP) 2 and 9 were significantly increased in ambient PM-treated VSMCs, with MMP9 levels being significantly higher in WPM-treated VSMCs than in those treated with SPM. As expected, migration was significantly increased in all tested PAHs (anthracene, ANT; benz(a)anthracene, BaA) and their oxygenated derivatives (9,10-Anthraquinone, AQ; 7,12-benz(a)anthraquinone, BAQ, respectively). The phosphorylated levels of focal adhesion kinase (FAK) and formation of the focal adhesion complex were significantly increased in ambient PM or PAH-treated VSMCs, and these effects were blocked by administration of NAC or α-NF, an inhibitor of AhR, the receptor that allows PAH uptake. Subsequently, the levels of phosphorylated Src and NRF, the downstream targets of FAK, were altered with a pattern similar to that of p-FAK. CONCLUSIONS: PAHs, including oxy-PAHs, in ambient PM may have dual effects that lead to an increase in VSMC migration. One is the generation of oxidative stress followed by MMP upregulation, and the other is actin reorganization that results from the activation of the focal adhesion complex.

LRP5 Regulates HIF-1α Stability via Interaction with PHD2 in Ischemic Myocardium.

Low-density lipoprotein receptor-related protein 5 (LRP5) has been studied as a co-receptor for Wnt/ß-catenin signaling. However, its role in the ischemic myocardium is largely unknown. Here, we show that LRP5 may act as a negative regulator of ischemic heart injury via its interaction with prolyl hydroxylase 2 (PHD2), resulting in hypoxia-inducible factor-1α (HIF-1α) degradation. Overexpression of LRP5 in cardiomyocytes promoted hypoxia-induced apoptotic cell death, whereas LRP5-silenced cardiomyocytes were protected from hypoxic insult. Gene expression analysis (mRNA-seq) demonstrated that overexpression of LRP5 limited the expression of HIF-1α target genes. LRP5 promoted HIF-1α degradation, as evidenced by the increased hydroxylation and shorter stability of HIF-1α under hypoxic conditions through the interaction between LRP5 and PHD2. Moreover, the specific phosphorylation of LRP5 at T1492 and S1503 is responsible for enhancing the hydroxylation activity of PHD2, resulting in HIF-1α degradation, which is independent of Wnt/ß-catenin signaling. Importantly, direct myocardial delivery of adenoviral constructs, silencing LRP5 in vivo, significantly improved cardiac function in infarcted rat hearts, suggesting the potential value of LRP5 as a new target for ischemic injury treatment.

Oxygenated polycyclic aromatic hydrocarbons from ambient particulate matter induce electrophysiological instability in cardiomyocytes.

BACKGROUND: Epidemiologic studies have suggested that elevated concentrations of particulate matter (PM) are strongly associated with an increased risk of developing cardiovascular diseases, including arrhythmia. However, the cellular and molecular mechanisms by which PM exposure causes arrhythmia and the component that is mainly responsible for this adverse effect remains to be established. In this study, the arrhythmogenicity of mobilized organic matter from two different types of PM collected during summer (SPM) and winter (WPM) seasons in the Seoul metropolitan area was evaluated. In addition, differential effects between polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs (oxy-PAHs) on the induction of electrophysiological instability were examined. RESULTS: We extracted the bioavailable organic contents of ambient PM, measuring 10 µm or less in diameter, collected from the Seoul metropolitan area using a high-volume air sampler. Significant alterations in all factors tested for association with electrophysiological instability, such as intracellular Ca2+ levels, reactive oxygen species (ROS) generation, and mRNA levels of the Ca2+-regulating proteins, sarcoplasmic reticulum Ca2+ATPase (SERCA2a), Ca2+/calmodulin-dependent protein kinase II (CaMK II), and ryanodine receptor 2 (RyR2) were observed in cardiomyocytes treated with PM. Moreover, the alterations were higher in WPM-treated cardiomyocytes than in SPM-treated cardiomyocytes. Three-fold more oxy-PAH concentrations were observed in WPM than SPM. As expected, electrophysiological instability was induced higher in oxy-PAHs (9,10-anthraquinone, AQ or 7,12-benz(a) anthraquinone, BAQ)-treated cardiomyocytes than in PAHs (anthracene, ANT or benz(a) anthracene, BaA)-treated cardiomyocytes; oxy-PAHs infusion of cells mediated by aryl hydrocarbon receptor (AhR) was faster than PAHs infusion. In addition, ROS formation and expression of calcium-related genes were markedly more altered in cells treated with oxy-PAHs compared to those treated with PAHs. CONCLUSIONS: The concentrations of oxy-PAHs in PM were found to be higher in winter than in summer, which might lead to greater electrophysiological instability through the ROS generation and disruption of calcium regulation.

Low density lipoprotein receptor-related protein 1 regulates cardiac hypertrophy induced by pressure overload.

BACKGROUND: Cardiac hypertrophy is associated with functional changes in cardiomyocytes, which often results in heart failure. The low-density lipoprotein receptor-related protein 1 (LRP1) is a large multifunctional endocytic receptor involved in many physiological and pathological processes. However, its function in the development of cardiac hypertrophy remains largely unclear. METHODS: Adenoviral constructs were used for either overexpression or silencing of LRP1 in both in vitro and in vivo experiments. Cardiac function was measured using the Millar catheter. RESULTS: LRP1 expression was upregulated in both transverse aortic constriction (TAC)-induced hypertrophic myocardium and catecholamine (phenylephrine (PE) and norepinephrine (NE))- and angiotensin II (AngII)-induced hypertrophic cardiomyocytes. In addition, cell surface area, protein/DNA ratio, and the mRNA levels of hypertrophic markers were significantly increased in LRP1-overexpressing cardiomyocytes without catecholamine stimulation. Conversely, LRP1 inhibition by LRP1-specific siRNA or a specific ligand-binding antagonist (RAP) significantly rescued hypertrophic effects in PE, NE, or AngII-induced cardiomyocytes. LRP1 overexpression induced PKCα, then activated ERK, resulting in cardiac hypertrophy with the downregulation of SERCA2a and calcium accumulation, which was successfully restored in both LRP1-silenced cardiomyocytes and TAC-induced hearts. CONCLUSIONS: LRP1 regulates cardiac hypertrophy via the PKCα-ERK dependent signaling pathway resulting in the alteration of intracellular calcium levels, demonstrating that LRP1 might be a potential therapeutic target for cardiac hypertrophy.

Effects of matrix metalloproteinase 13 on vascular smooth muscle cells migration via Akt-ERK dependent pathway.

Migration of vascular smooth muscle cells (VSMCs) is an early event of atherosclerosis, which is mediated mainly by matrix metalloproteinase (MMP) 2 and 9. Because MMP13 is associated with tumor cells migration, we hypothesized that MMP13 participates in VSMC migration induced by certain stimuli such as platelet-derived growth factor (PDGF) and angiotensin II (Ang II). We found that the mRNA level of MMP13 in rat aortic smooth muscle cells (RAoSMCs) was increased by both PDGF and Ang II. We observed the significant decrease of migration in PDGF- or Ang II-treated RAoSMCs by MMP13 specific inhibitor treatment. Silencing of MMP13 by a specific small interfering RNA (siRNA) significantly decreased expression of the active form of MMP13, which is followed by the decreased migration of PDGF- or Ang II-treated RAoSMCs. Interestingly, we observed synergistic inhibitory effects on migration by treatment with MMP2 and 13 or MMP9 and 13 inhibitors compared with that in single treatments. Moreover, we found that cordycepin, a known inhibitor of VSMC migration, caused significant downregulation of MMP2, 9, and 13 expression in PDGF-treated RAoSMCs. We further show that the expression level of MMP13 was significantly decreased by the treatment of Akt or ERK specific inhibitor in PDGF-treated RAoSMCs. Together, our data strongly suggest that MMP13 involves VSMCs migration via an Akt and ERK-dependent regulation [corrected].