Ginkgo biloba extract inhibits oxidized low-density lipoprotein (oxLDL)-induced matrix metalloproteinase activation by the modulation of the lectin-like oxLDL receptor 1- regulated signaling pathway in human umbilical vein endothelial cells  Kun-Ling Tsai, PhD, Yuh-Lih Chang, PhD, Po-Hsun Huang, PhD, Yung-Hsin Cheng, PhD, Ding-Hao Liu, MD, Hsiao-Yun Chen, MS, Chung-Lan Kao, MD, PhD  Journal of Vascular Surgery  Volume 63, Issue 1, Pages 204-215.e1 (January 2016) DOI: 10.1016/j.jvs.2014.05.098 Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 1 Effect of Gingko biloba extract (GbE) on oxidized low-density lipoprotein (oxLDL)-induced matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3 activation and tissue inhibitor of MMP-1 (TIMP-1) expression. A, MMP-1, MMP-2, MMP-3 and (B) TIMP-1 messenger RNA (mRNA) or (C-E) protein levels in human umbilical vein endothelial cells (HUVECs) that were pretreated with GbE (12.5-100 μM) for 2 hours, followed by exposure to oxLDL (130 μg/mL) for an additional 24 hours. F, Enzyme linked immunosorbent assay (ELISA) assay and (G) MMP-1, MMP-2, and MMP-3 activity assays were used to test MMP-1, MMP-2, and MMP-3 release and activity under oxLDL stimulation. Cells were lysed at the end of the incubation period, and lectin-like oxLDL receptor 1 (LOX-1) mRNA and protein were analyzed by real-time polymerase chain reaction and Western blotting, respectively. The mRNA and protein levels of MMP-1, MMP-2, and MMP-3, and TIMP-1 were normalized to the level of β-actin. The data illustrated in the graphs represent the mean ± standard error of the mean of three different experiments. #P < .05 vs untreated control; *P < .05 vs oxLDL treatment. Journal of Vascular Surgery 2016 63, 204-215.e1DOI: (10.1016/j.jvs.2014.05.098) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 2 Effect of Gingko biloba extract (GbE) on oxidized low-density lipoprotein (oxLDL)-induced lectin-like oxLDL receptor 1 (LOX-1) activation. Human umbilical vein endothelial cells (HUVECs) were pretreated with GbE (12.5-100 μM) or diphenyleneiodonium chloride (DPI; 5 μM) for 2 hours, followed by exposure to oxLDL (130 μg/mL) for 24 hours. Cells were lysed at the end of the incubation period, and (A) LOX-1 messenger RNA (mRNA) and (B and C) protein were analyzed by real-time polymerase chain reaction and Western blotting, respectively. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. The mRNA and protein levels of LOX-1 were normalized to the level of β-actin. The data illustrated in the graph represent the mean ± standard error of the mean of three different experiments. #P < .05 vs untreated control; *P < .05 vs oxLDL treatment. Journal of Vascular Surgery 2016 63, 204-215.e1DOI: (10.1016/j.jvs.2014.05.098) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 3 Inhibitory effects of Gingko biloba extract (GbE) on oxidized low-density lipoprotein (oxLDL)-induced reactive oxygen species (ROS) generation in HUVECs. After preincubation for 2 hours with the lectin-like oxLDL receptor 1 (LOX-1) antibody (ab), GbE (12.5-100 μM), and ROS inhibitor (diphenyleneiodonium chloride [DPI]), cells were treated with 130 μg/mL oxLDL for 2 hours, followed by a 1-hour incubation with the superoxide-sensitive fluorescent probe dihydroethidium (DHE; 10 μM). A, Fluorescence images exhibited the ROS level in control cells and human umbilical vein endothelial cells (HUVECs) stimulated with oxLDL in the presence GbE or LOX-1 antibody. (B) The fluorescence intensity of cells was measured with a fluorescence microplate reader. The fluorescence distribution of DHE oxidation is expressed as a percentage of increased intensity. mAb, monoclonal antibody. The activity of (C) superoxide dismutase (SOD) and (D) catalase in HUVECs stimulated with oxLDL in the absence or presence of indicated concentrations of GbE was determined. The data illustrated in the graph represent the mean ± standard error of the mean of three different experiments. #P < .05 vs untreated control; *P < .05 vs oxLDL treatment. Journal of Vascular Surgery 2016 63, 204-215.e1DOI: (10.1016/j.jvs.2014.05.098) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 4 Inhibitory effect of Gingko biloba extract (GbE) on oxidized low-density lipoprotein (oxLDL)-induced protein kinase C (PKC) activation. Human umbilical vein endothelial cells (HUVECs) were preincubated for 2 hours with GbE (12.5-100 μM) and the reactive oxygen species (ROS) inhibitor (diphenyleneiodonium chloride [DPI]), followed by exposure to oxLDL (130 μg/mL) for 2 hours. A and B, At the end of the incubation period, the levels of phosphorylated (p)-PKC were determined by immunoblotting. The protein levels of p-PKC-α and PKC-β were normalized to the level of total PKC. ab-LOX-1, antibody to lectin-like oxLDL receptor 1. C, PKC-α activity in whole-cell lysates was measured by a fluorescein green assay kit. The data illustrated in the graph represent the mean ± standard error of the mean of three different experiments. #P < .05 vs untreated control; *P < .05 vs oxLDL treatment. Journal of Vascular Surgery 2016 63, 204-215.e1DOI: (10.1016/j.jvs.2014.05.098) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 5 Protective effect of Gingko biloba extract (GbE) on oxidized low-density lipoprotein (oxLDL)-activated expression of extracellular signal-regulated kinase (ERK) and phosphorylated (p)-ERK. Human umbilical vein endothelial cells (HUVECs) were preincubated for 2 hours with GbE (12.5-100 μM) and the reactive oxygen species (ROS) inhibitor (diphenyleneiodonium chloride [DPI]), followed by exposure to oxLDL (130 μg/mL) for 1 hour. A and B, At the end of the incubation period, the levels of p-ERK were determined by immunoblotting. The protein level of p-ERK was normalized to the level of total ERK. The data illustrated in the graph represent the mean ± standard error of the mean of three different experiments. #P < .05 vs untreated control; *P < .05 vs oxLDL treatment. Journal of Vascular Surgery 2016 63, 204-215.e1DOI: (10.1016/j.jvs.2014.05.098) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 6 Oxidized low-density lipoprotein (oxLDL)-induced activation of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB in the presence of pharmacologic inhibitors or Gingko biloba extract (GbE). The pretreatment of human umbilical vein endothelial cells (HUVECs) with the indicated concentrations of diphenyleneiodonium chloride (DPI), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), Gö6976, PD98059, or GbE attenuated ERK activation, NF-κB inhibitor-α (I-κBα) degradation, and NF-κB activation induced by oxLDL. HUVECs were pretreated with each inhibitor or GbE for 2 hours, followed by incubation with oxLDL (130 μg/ml) for 1 hour. A-D, The cells were lysed at the end of the incubation period, and the proteins were analyzed by Western blotting. The protein levels of phosphorylated (p)-ERK, NF-κB, and IκB-α were normalized to the levels of total ERK, proliferating cell nuclear antigen (PCNA), and β-actin, respectively. The data illustrated in the graph represent the mean ± standard error of the mean of three separate experiments. #P < .05 vs untreated control; *P < .05 vs oxLDL treatment. Journal of Vascular Surgery 2016 63, 204-215.e1DOI: (10.1016/j.jvs.2014.05.098) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 7 Gingko biloba extract (GbE) reversed oxidized low-density lipoprotein (oxLDL)-diminished peroxisome proliferator-activated receptor-γ (PPAR-γ) expression. A and B, Human umbilical vein endothelial cells (HUVECs) were preincubated for 2 hours with GbE (12.5-100 μM) or the reactive oxygen species (ROS) inhibitor (diphenyleneiodonium chloride [DPI]), antibody to lectin-like oxLDL receptor 1 (ab-LOX-1), protein kinase C (PKC) inhibitor (Gö6976), extracellular signal-regulated kinase (ERK) inhibitor (PD98069), and nuclear factor (NF)-κB inhibitor (pyrrolidine dithiocarbamate [PDTC]), (C and D) followed by exposure to oxLDL (130 μg/mL) for 1 hour. At the end of the incubation period, the levels of PPAR-γ for both were determined by immunoblotting. The protein levels of PPAR-γ were normalized to the level of β-actin. The data illustrated in the graph represent the mean ± standard error of the mean of three different experiments. #P < .05 vs untreated control; *P < .05 vs oxLDL treatment. Journal of Vascular Surgery 2016 63, 204-215.e1DOI: (10.1016/j.jvs.2014.05.098) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 8 Inhibition of peroxisome proliferator-activated receptor-γ (PPAR-γ) with short interfering (si)RNA antagonized the effects of Gingko biloba extract (GbE) on oxidized low-density lipoprotein (oxLDL)-induced matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3 activation. Human umbilical vein endothelial cells (HUVECs) were transfected with PPAR-γ siRNA for 72 hours and were treated with 100 μg/mL GbE for 1 hour, followed by exposure to 130 mg/mL oxLDL for 24 hours. A, The Western blot analysis of the si-5' adenosine monophosphate-activated protein kinase-a knockdown efficiency. B and C, The cell lysates were analyzed by Western blot using anti-MMP-1, anti-MMP-2, and anti-MMP-3, or anti-β-actin antibodies. mRNA, Messenger RNA. D, Enzyme-linked immunosorbent assay (ELISA) was used to examine the protective effects of GbE on oxLDL-promoted MMP-1, MMP-2, and MMP-3 release. E, Inhibitory efficiency of GbE and quercetin were compared in the same concentration. The values represent means ± standard error of the mean from four separate experiments. #P < .05 vs untreated control; *P < .05 vs oxLDL treatment; &P < .05 vs GbE + oxLDL treatment. Journal of Vascular Surgery 2016 63, 204-215.e1DOI: (10.1016/j.jvs.2014.05.098) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 9 Schematic diagram shows the signaling cascades involved in the attenuation of matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3 expression in cells exposed to oxidized low-density lipoprotein (oxLDL) that were treated with Gingko biloba extract (GbE). As depicted, GbE inhibited oxLDL-induced lectin-like oxLDL receptor 1 (LOX-1) activation, thereby inhibiting oxLDL-facilitated reactive oxygen species (ROS) formation and protein kinase C (PKC) activation. We also confirmed that Ca++, PKC, mitogen-activated protein kinase (MAPK), and ROS were involved in the protective actions of GbE. Moreover, we revealed that GbE pretreatment reversed oxLDL-repressed peroxisome proliferator-activated receptor-γ (PPAR-γ) expression. Finally, we found that GbE mitigates oxLDL-promoted MMP-1, MMP-2, and MMP-3 activation by modulation of PPAR-γ. The → indicates activation or induction, and ⊢indicates inhibition or blockade. Journal of Vascular Surgery 2016 63, 204-215.e1DOI: (10.1016/j.jvs.2014.05.098) Copyright © 2016 Society for Vascular Surgery Terms and Conditions