Introduction
In the early stages of atherogenesis, the adhesiveness of vascular endothelium to monocytes is increased, which may be mediated by endothelial expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and others as a result of endothelial dysfunction [
1]. Clinical evidence suggests that hyperglycemia is an independent risk factor of diabetes-associated atherosclerosis [
2]. In vitro studies also suggest that high concentration of glucose (high glucose) could induce ICAM-1 expression [
3] and increase the production of chemokines such as interleukin-6 (IL-6) through a tyrosine kinase mechanism involving the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways [
4]. However, the detailed mechanisms by which high glucose could induce endothelial adhesiveness have not been fully clarified.
It was suggested that leukocyte-endothelial interaction may be augmented by high glucose or hyperglycemia in a NF-kB-dependent fashion [
5]. It was also reported that high glucose might induce ICAM-1 accumulation by a non-specific osmotic effect [
6]. On the other hand, VCAM-1 expression was shown to increase in endothelial cells by serum from type 1 diabetic patients but not by high-glucose stimulation [
7]. Previous studies further indicated that the short-term presence of inflammatory cytokines rather than high glucose could induce endothelial expression of VCAM-1 and ICAM-1 [
8]. Accordingly, the mechanisms of high-glucose-induced endothelial expression of adhesion molecules could be complex. It was not known whether and how inflammatory cytokines could alter endothelial expression of adhesion molecules in the presence of high glucose.
Ginkgo biloba extract (GBE), an herbal medicine with antioxidant mechanisms, has been used as a therapeutic agent for some cardiovascular and neurological disorders [
9]. Although the exact mechanism has not been completely clarified, cumulative
in vitro and
in vivo evidence suggests the protective effects of GBE in ischemia/reperfusion injury [
10] by reducing oxidative stress [
11]. Our previous studies also showed that GBE may inhibit neointimal hyperplasia [
12] and cytokine-stimulated endothelial adhesiveness to monocytes [
13], at least in part by inducing heme oxygenase-1 to reduce intracellular oxidative stress [
14]. However, it was not known whether and how GBE could reduce high-glucose-induced endothelial adhesiveness.
Clinical evidence suggests that endothelial adhesion molecule expression is enhanced in the aorta and the internal mammary artery of diabetic patients [
15]. Additionally, hyperglycemia-mediated adhesion of neutrophils to rat endothelial cells could be attenuated by superoxide dismutase
in vivo[
16]. These findings support the hypothesis that intracellular oxidative stress might be critical to high-glucose-mediated, diabetes-associated atherosclerosis. The current study was conducted 1) to clarify whether and how redox-related mechanisms could contribute to high-glucose-induced endothelial adhesiveness to monocytes, and 2) to investigate whether GBE as an herbal antioxidant could reduce
in vitro endothelial adhesiveness in the presence of high glucose. Our findings may help to further clarify the complex mechanisms by which high glucose might induce endothelial inflammation and to provide the rationale to the potential role of anti-inflammatory strategy for vascular protection in clinical hyperglycemia.
Materials and methods
This in vitro study was approved by the research committee of Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China.
Reagents
Endothelial cell growth medium (M200) was obtained from Cascade Biologics (Cascade Biologics, Portland, OR). The GBE (Cerenin) was purchased from Dr. Willmar Schwabe (Dr. Willmar Schwabe, Inc.). 2′,7′-bis(2-carboxyethyl)-5(6)- carboxyfluorescein acetoxymethyl ester (BCECF-AM) and 2′,7′- dichlorofluorescein diacetate (DCFH-DA) were obtained from Molecular Probes (Invitrogen/Molecular Probes). Antibodies against human ICAM-1 and β-actin were obtained from R&D Systems (R&D Systems) and Chemicon (Chemicon, Temecula), respectively. Unless otherwise specified, all chemicals and reagents were from Sigma (Sigma).
Cell cultures and cell viability assay
HAECs (Cascade Biologics) and THP-1 from American Type Culture Collection (Rockville) were cultured as described previously [
14]. After incubation with glucose, mannitol, or GBE, cell viability was always found to be greater than 95% by using the trypan blue exclusion method or a 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma).
Monocyte-endothelial cell adhesion assay
The adherence of THP-1, a human monocytic cell line, to glucose- and mannitol-activated HAECs was examined under static conditions to explore the endothelial cell–leukocyte interaction. Adhesion assays were performed as described previously [
14]. HAECs were grown to sub-confluence in 24-well plates and treated with glucose or mannitol for 3 days, followed by treatment with GBE or NAC for 1 day.
Western blot analysis
Western blot analyses were performed using the methods as described previously [
14].
Enzyme-linked immunosorbent assay (ELISA)
HAECs were continuously incubated in a high-glucose medium with or without antioxidants for 4 days. The levels of secreted IL-6 in the medium were determined by ELISA with the human IL-6 kit (BioSource). The procedures were carried out according to the instructions of the manufacturer.
Electrophoretic mobility shift assay (EMSA)
EMSA was performed as previously described [
13]. The synthetic, double-stranded oligonucleotides used in the gel shift assay as the STAT1 and STAT3 probes were 5′-GAT CTT CAG TTT CAT ATT ACT CTA AAT CCA GGA TC-3′ and 5′-GAT CCC TTC GGG AAT TCC TAG ATC-3′, respectively. For supershift analyses, anti-STAT1 and anti-STAT3 monoclonal antibodies (Santa Cruz) were added to the reaction mixture 60 min before the addition of labeled oligonucleotides at 4°C.
Measurement of ROS production
ROS production in HAECs was determined by a fluorometric assay using DCFH-DA as a probe to detect the presence of H2O2. The fluorescence intensity (RFU) was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm by using a fluorescent microplate reader (VICTPR2 Multilabel Readers, USA).
Statistical analysis
All data were expressed as the mean ± SEM. Intergroup comparisons were performed by using a Student’s t test or one-way analysis of variance (ANOVA). A p value < 0.05 was considered statistically significant.
Discussion
This study reveals several major findings. First, treatment of HAECs with high glucose for 4 days significantly increased endothelial ICAM-1 expression, which in turn enhanced endothelial adhesiveness to monocytes. Second, long-term presence of high glucose stimulated endothelial IL-6 secretion via the redox-dependent mechanism, which may then induce STAT3 activation and consequent ICAM-1 expression. Third, in parallel to high glucose, mannitol exerted the similar effects on inducing endothelial adhesiveness. Fourth, pretreatment with GBE inhibited high-glucose- as well as mannitol-induced intracellular ROS production, IL-6 secretion, STAT3 activation, ICAM-1 accumulation, and consequent endothelial adhesiveness to monocytes. Fifth, in parallel to GBE, NAC also exerted the similar endothelial protection effects in the presence of high glucose or mannitol. Taken together, it is suggested that long-term presence of high glucose might induce STAT3-mediated ICAM-1-dependent endothelial adhesiveness to monocytes, which requires the osmotic-related redox-dependent IL-6 activation. Furthermore, GBE, an antioxidant herb medicine, could prevent high-glucose-induced endothelial inflammation mainly by inhibiting IL-6 activation.
Hyperglycemia is considered one of the major pathogenic factors for atherogenesis and the progression of atherosclerosis in diabetes mellitus [
23]. However, the detailed molecular pathological mechanisms may be varied according to the different findings in previous
in vitro and
in vivo studies. In the current study, endothelial ICAM-1 expression was increased by chronic treatment with high glucose, resulting in the upregulation of endothelial adhesiveness to monocytes. Our findings agreed in part with the previous suggestion that ICAM-1 may play an essential role in mediating endothelial adhesiveness to monocytes [
24]. Besides, in the present study, long-term presence of high glucose increased endothelial IL-6 expression at least in part via the oxidative related mechanisms, which may then contribute to the increase of endothelial adhesiveness to monocytes. These findings also agreed in part with that of previous studies with different study designs, in which IL-6 was shown to induce endothelial ICAM-1 expression [
25] and oxidative stress could be involved in the induction of IL-6 and ICAM-1 in HAECs treated with intermittent high glucose [
26]. Finally, in the current study, both high glucose and mannitol could induce endothelial IL-6 expression, which supports the previous suggestion that high glucose may increase endothelial ICAM-1 expression by a nonspecific osmotic effect [
6]. More interestingly, such effects on endothelial ICAM-1 expression could be reversible and directly related to the presence of high glucose since it could disappear gradually after high-glucose medium was replaced by normal medium (please see
Additional file 1: Figure S3). Most mammalian cells respond to changes in cellular volume with a net movement of water driven by a redistribution of salt and or small organic molecules [
27,
28]. However, it is still not known which receptor or sensor for osmotic effect may regulate cell volume as well as cell signal transduction in endothelial cell. One of the possible receptors is receptor tyrosine kinase receptors (RTKs). Growth control and apoptosis in mammalian cells are highly regulated by RTKs. A number of reports also implicated a role of RTKs in osmosensing and volume control as upstream regulators. That osmotic stress activates p38 MAPK and JNK were described in mammalian cells [
29‐
31]. Accordingly, our study connected the complex findings of previous studies and helped to elucidate a whole picture of detailed mechanisms for high-glucose-induced endothelial adhesiveness that mimics early
in vivo atherogenesis.
It was recently shown that Interleukin-6 (IL-6) plays a beneficial role in anti-inflammatory activity to against bacterial infections [
32], and enhances insulin action immediately at early recovery [
33,
34]. However, it is well known that IL-6 is a multifunctional cytokine regulating cellular responses especially in inflammation. It has been suggested that diabetes-related oxidative stress may induce vascular IL-6 expression and activate IL-6R and gp130, which in turn activates the JAK2/STAT3 signaling cascade [
35]. In the present study, STAT1 and STAT3 rather than AP-1 and NF-kB were activated by high glucose in HAECs. More importantly, piceatannol, a STAT1/3 inhibitor, but not fludarabine, a STAT1 inhibitor, significantly suppressed high-glucose-induced ICAM-1 expression in HAECs, suggesting the contribution of STAT3 pathways in ICAM-1 induction. It has been previously shown that IL-6 may activate endothelial STAT3 and ICAM-1 expression [
25]. The present study further indicated that long-term presence of high glucose may induce endothelial IL-6 production by the osmotic-related redox-dependent mechanisms, which in turn activate endothelial ICAM-1 expression mainly via the STAT3 transcriptional pathways. Our findings support the rationale that strict glucose control may be critical to reduce osmotic injury and related oxidative stress for endothelial protection in clinical hyperglycemia.
There have been very few, mainly
in vitro, evidence for the effects of GBE in the presence of high glucose [
36]. To our knowledge, this is the first study demonstrating the direct endothelial protection effects of GBE in the presence of long-term high glucose. In this study, pretreatment of GBE could does-dependently suppressed HG-induced ICAM-1 accumulation in HAECs. (
Additional file 1: Figure S2) It was also shown that NAC, similar to GBE, could also reduce high-glucose-induced ROS generation, IL-6 secretion, and ICAM-1 accumulation in HAECs. Taken together, our findings suggest that GBE, as an antioxidant herb medicine, may inhibit IL-6 activation and consequent ICAM-1 expression probably by its antioxidant as well as anti-inflammatory effect. It was recently shown that GBE may have anti-oxidant ability by its free radical scavenger property [
37] and mitochondrial uncoupling effect [
38]. However, high dosage GBE (50–200 μg/mL) didn’t reduce hydrogen peroxide generation by mitochondria in rats heart [
39], which may be due to the complex components of GBE and its selective uncoupling of mitochondrial oxidative phosphorylation. Future studies are required to validate the current findings in
in vivo hyperglycemia.
The detailed mechanisms of the anti-inflammatory effects of GBE for vascular protection might be complex and varied in different experimental as well as clinical conditions. We have recently shown the contribution of Nrf-2-mediated heme oxygenase 1 induction to the inhibitory effects of GBE on TNF-α-induced VCAM-1 expression in endothelial cells [
14]. In the present study, GBE was shown to inhibit high-glucose-induced ICAM-1 expression and consequent adhesiveness of HAECs. These findings are in line with our previous findings that GBE could inhibit cytokine-stimulated endothelial adhesiveness [
13], vascular smooth muscle cell proliferation [
40], and
in vivo neointimal hyperplasia [
12] mainly by its inhibitory effects on intracellular oxidative stress. Though the molecular mechanisms could be different, GBE may have similar anti-inflammatory effects in either TNF-α-induced or high-glucose-induced endothelial adhesiveness, suggesting the potential universal role of GBE in endothelial protection. However, given the complex and multiple effects of GBE shown in the current and previous studies [
12‐
14], it is possible that other redox-independent mechanisms may be also involved in the beneficial effects of GBE on endothelial protection in the presence of high glucose. It might be important since the pathological changes are much more complex in
in vivo hyperglycemia. Future
in vivo studies are required to clarify this issue.
It should be indicated that GBE used in the current and our previous studies is the same preparation with fixed portions of standard components [
12‐
14]. Though the individual effects of the major components of GBE, flavone glycoside and terpenlactones, on atherogenesis and vascular inflammation remain to be investigated, the dosage of GBE used in the current study is similar to or equivalent to that had been used in our previous
in vitro and
in vivo studies [
12‐
14]. Furthermore, it was shown in the previous study that in the volunteers, after administration of GBE (160 mg) in free (Ginkgoselect) or phospholipid complex (Ginkgoselect Phytosome) forms, the maximum plasma concentrations, C(max), of total ginkgolides A, B and bilobalide were 85.0 and 181.8 ng/mL for Ginkgoselect and Ginkgoselect Phytosome, respectively. The C(max) values reached at 120 minutes for the free form and at 180–240 minutes for the phospholipid complex form [
41]. In such case, the serum level seems similar to the concentration used in the current and previous studies. As that had been mentioned previously, the dose of GBE used in the current and previous studies may not be directly translated to that in clinical situations since there might be significant variety in individual drug metabolism and accumulation effects of such kind of herb medicine in different person [
12,
13]. However, the GBE and NAC used in the current study have also been widely and safely used clinically for years. Future
in vivo studies or even clinical trials seem feasible to test the potential role and investigate the proper dose of GBE and other antioxidant/anti-inflammatory agents such as NAC for vascular protection especially in patients with chronic hyperglycemia and elevated serum IL-6 levels.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Jia-Shiong Chen conducted the experiments and contributed to the study implementation, statistical analysis, interpretation, and the preparation of the manuscript. Yung-Hsiang Chen conducted the experiments and contributed to the study conception and design, implementation, and the preparation of the manuscript. Both JS Chen and YH Chen contributed equally to this paper. Po-Hsun Huang and Hsiao-Ya Tsai helped to conduct the experiments and contributed to the study conception and design, implementation, and interpretation. Yuh-Lien Chen contributed to the study conception and design, implementation, and interpretation. Shing-Jong Lin contributed to the study conception and design. Jaw-Wen Chen supervised the study conduction and contributed to the study conception and design, implementation, statistical interpretation, the preparation and finalization of the manuscript. All authors approved the final manuscript for publication.