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Cell Biology International (2012) 36, 511–517 (Printed in Great Britain)
Effects of ghrelin on homocysteine-induced dysfunction and inflammatory response in rat cardiac microvascular endothelial cells
Dongjuan Wang*1, Haichang Wang*1, Peng Luo†1, Andrew Hwang‡, Dongdong Sun*, Yabin Wang*, Zheng Zhang*, Nan Liu*, Shenxu Wang*, Chengxiang Li* and Feng Cao*2
*Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi, 710032, †Department of Cardiovascular surgery, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi, 710032, China, and ‡Department of Radiology, Molecular Imaging Program of Stanford, Stanford University, Stanford, CA, USA


Ghrelin is a well-characterized hormone that has protective effects on endothelial cells. Elevated HCY (homocysteine) can be a cardiovascular risk factor, but it is not known whether ghrelin can inhibit HCY-induced dysfunction and inflammatory response in rat CMECs (cardiac microvascular endothelial cells). We found that HCY treatment for 24 h inhibited proliferation and NO (nitric oxide) secretion, but with increased cell apoptosis and secretion of cytokines in CMECs. In contrast, ghrelin pretreatment significantly improved proliferation and NO secretion, and inhibited cell apoptosis and secretion of cytokines in HCY-induced CMECs. In addition, Western blot assay showed that NF-κB (nuclear factor κB) and cleaved-caspase 3 expression were elevated, and PCNA (proliferating cell nuclear antigen) and eNOS (endothelial nitric oxide synthase) expression were decreased after treatment with HCY, which was significantly reversed by pretreatment with ghrelin. The data suggest that ghrelin inhibits HCY-induced CMEC dysfunction and inflammatory response, probably mediated by inhibition of NF-κB activation.


Key words: cardiac microvascular endothelial cell (CMEC), ghrelin, homocysteine (HCY), inflammatory response, NF-κB

Abbreviations: AG, acylated ghrelin, BrdU, bromodeoxyuridine, CMEC, cardiac microvascular endothelial cell, CVD, cardiovascular disease, DAPI, 4′,6-diamidino-2-phenylindole, eNOS, endothelial nitric oxide synthase, GH, growth hormone, HCY, homocysteine, ICAM-1, intercellular adhesion molecule 1, IκB, inhibitory κB, IL, interleukin, MCP-1, monocyte chemoattractant protein 1, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, NAC, N-acetyl-K-cysteine, NF-κB, nuclear factor κB, NO, nitric oxide, PCNA, proliferating cell nuclear antigen, PI3K, phosphoinositide 3-kinase, SD, Sprague–Dawley, TUNEL, terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labelling, UAG, unacylated ghrelin

1These authors have contributed equally to this work.

2To whom correspondence should be addressed (email wind8828@gmail.com).


1. Introduction

CVD (cardiovascular disease) is one of the most dangerous threats to human health, and its incidence and mortality rates are among the highest of most diseases. Dysfunction of CMECs (cardiac microvascular endothelial cells) is closely related to CVDs, including diabetic cardiomyopathy, heart failure and microvascular angina (Avogaro et al., 2007; Kang and Yang, 2007). Our recent findings also suggested that endothelial cell dysfunction was an important pathophysiological event in ischaemic reperfusion injury, which involved microvascular endothelial cell dysfunction, swelling and apoptosis, and local ischaemia (Wei et al., 2010a).

Several epidemiological studies (de Ruijter et al., 2009; Oudi et al., 2010) have identified moderately elevated concentrations of HCY (homocysteine) as an independent risk factor for coronary heart disease, which may contribute to the development of atherosclerosis (McDowell and Lang, 2000). HCY could influence endothelial function, leading to a prothrombotic environment, platelet activation and endothelial leucocyte interactions (Pasterkamp et al., 2002). In addition, HCY acts as an atherogenic factor by promoting differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions (Zhang et al., 2009). Ghrelin is a 28-amino-acid peptide hormone produced principally in the stomach, which induces the release of GH (growth hormone) and stimulates food intake, regulates energy balance and induces adiposity (Nakazato et al., 2001, Tschop et al., 2000). Ghrelin has a variety of GH releasing-independent cardiovascular activities, such as promoting angiogenesis, reducing ischaemic reperfusion injury, enhancing vasodilation and alleviating heart failure (Kojima et al., 1999; Xu et al., 2005). In particular, ghrelin could inhibit inflammatory response and apoptosis in endothelial cells (Li et al., 2004).

However, the effects of ghrelin on HCY-induced CMECs and the molecular mechanisms responsible for these effects remain unclear. We have therefore investigated the effects of ghrelin on HYC-induced CMEC dysfunction and inflammatory response, and explored the mechanism involved.

2. Materials and methods

2.1. Animals

Twenty male SD (Sprague–Dawley) rats (100–150 g) were purchased from the Experimental Animal Center of the Fourth Military Medical University. All animal procedures were performed in accordance with protocols approved by the Fourth Military Medical University Animal Research Committee and International Research Animal Care Guidelines.

2.2. Isolation, cultivation and identification of CMECs

CMECs were isolated from SD rat hearts by enzyme dissociation (Nishida et al., 1993). Briefly, hearts were removed from male rats under aseptic conditions. After removal of the atria, right ventricle, visible connective tissue and valvular tissue, the left ventricle was immersed in 75% ethanol for 15 s to devitalize endocardial and epicardial endothelial cells. The tissue was minced and digested (0.02% collagenase type II for 6 min and 0.025% trypsin for 5 min at 37°C in a shaking bath), followed by filtration through a 100 μm nylon mesh and centrifugation at 2000 rev./min for 10 min. The pellet was resuspended with DMEM (Dulbecco's modified Eagle's medium) supplemented with 15% FBS (fetal bovine serum; Hyclone) at 37°C. The medium was refreshed every 3 days.

Cultured CMECs were identified by morphological and functional characteristics (Wei et al., 2010b). Passage 2 and 3 cells were used for further studies. The cells displayed ‘flagstone' morphology, and tested positive in both Dil-Ac-LDL (low-density lipoprotein) intake assay and immunofluorescence staining of vWF (von Willebrand factor).

2.3. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay for cell proliferation

Cells were plated overnight in 96-well tissue culture plastic dishes at 1×104 cells/ml. Medium was pretreated with ghrelin at 0, 10, 100, or 1000 ng/ml for 1 h. HCY was added to the medium at 0.1, 0.25, 0.5 or 1 mmol/l for 24 h. Cells from each group were harvested, and 100 μl MTT (Sigma) was added into each well and incubated for 4 h. At the end of this treatment, the incubation medium was removed and formazan crystals were dissolved in 100 μl DMSO. MTT reduction was quantified by measuring the light absorbance at 490 nm.

2.4. BrdU (bromodeoxyuridine) incorporation assay

Cells were allocated into 4 groups: C, control group; G, 100 ng/ml ghrelin; H, 0.25 mmol/l HCY; G+H, 100 ng/ml ghrelin+0.25 mmol/l HCY. CMECs were grown on 22 mm coverslips, and after growing to 70% confluent, 1 μg/ml BrdU was added for 1 h. The cells were fixed for 30 min in 4% (w/v) paraformaldehyde at room temperature and DNA denatured with 2 M HCl for 40 min. BrdU was stained with mouse anti-BrdU antibody (1:200, Abcam) and anti-mouse rhodamine-conjugated secondary antibody (1:200, Invitrogen), followed by detecting with fluorescence microscope to determine BrdU incorporation.

2.5. Assessment of apoptosis of CMECs

A total of 1×105 CMECs were treated with ghrelin and HCY, at the concentrations noted above. Apoptosis was detected by TUNEL (terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labelling; in green) assay using a Cell Death Detection Kit (Roche). For detection of total nuclei, the slides were covered with the mounting medium containing DAPI (4′,6-diamidino-2-phenylindole). At least 5 slides per block were evaluated using fluorescence microscope. Results were expressed as the proportion of the TUNEL positive CMECs nuclei to the total CMECs in percentage.

2.6. NO (nitric oxide) measurement

The amount of NO production released by CMECs was determined by measuring the concentration of nitrite, a metabolite of NO, with a modified Griess reaction method (Nanjing Jiancheng Institute of Biological Engineering). Briefly, CMECs were seeded at 1×105 cells/well on a 6-well plate 1 day before the treatment. Adherent cells were pretreated with ghrelin for 1 h and then with HCY for a further 24 h. Finally, supernatant was collected and mixed with an equal volume of modified Griess reagent for the colorimetric assay. After 10 min incubation at room temperature, the concentration of the resultant chromophore was measured spectrophotometrically at 550 nm after enzymatic conversion of the supernatant nitrate to nitrite by nitrate reductase. The nitrite concentration in the samples was calculated from nitrite standard curves made from sodium nitrite using the same culture medium.

2.7. Measurement of pro-inflammatory cytokines and adhesion molecules

CMECs were seeded 1×105 cells/well on a 6-well plate 1 day before the treatment. Adherent cells were pretreated with ghrelin for 1 h and then with HCY for a further 24 h. The supernatants were collected and the levels of IL-6 (interleukin 6), IL-8, MCP-1 (monocyte chemoattractant protein 1) and ICAM-1 (intercellular adhesion molecule 1) measured by ELISA kits (Shenzhen Juying Institute of Biological Engineering). Recombinant peptides were used to construct standard. Absorbance of standards and samples was determined spectrophotometrically at 450 nm using a microplate reader. Results were plotted according to the linear portion of the standard curve.

2.8. Western blot analysis

Cells from each group were cultured and harvested at the indicated time. Cells were washed 3 times with cold PBS, and scraped off using an ice-cold lysis buffer. Proteins were prepared and separated on SDS/10% PAGE gels before being transferred electrophoretically to nitrocellulose membranes. After blocking with 5% (w/v) non-fat dried skimmed milk powder, the membranes were immunoblotted with the appropriate primary antibody at 4°C overnight. The membranes were washed and further incubated with the secondary antibody at 37°C for 60 min. Blots were visualized using an ECL (enhanced chemiluminescence) system (Amersham).

The following primary antibodies were used: rabbit anti-rat NF-κB (nuclear factor κB) p65 (1:1000, Santa Cruz), rabbit anti-rat PCNA (proliferating cell nuclear antigen; 1:1000, Santa Cruz), rabbit anti-rat eNOS (endothelial nitric oxide synthase; 1:200, Santa Cruz), rabbit anti-rat IL-6 (1:1000, Abcam), mouse anti-rat ICAM-1 (1:1000, Abcam), rabbit anti-rat cleaved-caspase 3 (1:200, Santa Cruz), and mouse anti-rat β-actin (1:2000, Cell Signaling technology). Secondary antibodies were HRP (horseradish peroxidase)-conjugated goat anti-rabbit IgG (1:5000, Santa Cruz) and rabbit anti-mouse (1:5000, Santa Cruz).

2.9. Statistical analysis

All values were presented as means±S.D. (of n independent experiments). All data (except Western blot density) were compared by ANOVA followed by Bonferroni correction for post hoc analysis. P<0.05 was considered statistically significant. All of the statistical tests were performed using GraphPad Prism software version 5.0 (GraphPad Software).

3. Results

3.1. Ghrelin pretreatment and CMECs proliferation

CMECs proliferation was assessed by MTT assay. HCY significantly blunted CMECs proliferation at a concentration of 0.1 mmol/l (0.27±0.02 versus 0.31±0.03, P<0.05), 0.25 mmol/l (0.25±0.04 versus 0.31±0.03, P<0.01), 0.5 mmol/l (0.18±0.15 versus 0.313±0.03, P<0.01) and 1 mmol/l (0.14±0.02 versus 0.31±0.03, P<0.01: Figure 1A). However, pretreatment with ghrelin (100 or 1000 ng/ml) markedly inhibited HCY-mediated cell death (0.28±0.02 versus 0.20±0.02, P<0.05; 0.283±0.03 versus 0.20±0.02, P<0.05). In addition, treatment of the cells with the antioxidant NAC (N-acetyl-K-cysteine) also significantly attenuated the suppressive effects of HCY on CMECs proliferation. Combined treatment with NAC and ghrelin further attenuated proliferation compared with NAC alone, indicating that HCY still inhibited proliferation when the effect of HCY autoxidation on CMECs was abrogated (Figure 1B).

PCNA was highly expressed in growing cells. Its expression in CMECs analysed by Western blotting showed that it was down-regulated by HCY (0.25 mmol/l) compared with the control group (P<0.05). In addition, ghrelin (100 ng/ml) markedly increased PCNA both in the basal (P<0.05) and HCY-induced groups (P<0.05) (Figures 1C and 1D).

The effect of ghrelin on the proliferation of CMECs was assayed by BrdU. Red nuclei detected (positive cells) were decreased by HCY (0.25 mmol/l), whereas ghrelin (100 ng/ml) pretreatment increased them (Figures 1E and 1F), demonstrating that pretreatment with ghrelin promoted CMEC proliferation.

3.2. Ghrelin's anti-apoptotic effect on CMECs

To verify whether HCY induced CMECs apoptosis could be reversed by ghrelin, the TUNEL assay was used to assess the level of apoptosis of CMECs. The percentage of apoptotic CMECs in the HCY (0.25 mmol/l) group was ∼2 times greater than in the control group (38.3±1.7% versus 15.6±2.0%, P<0.05; Figure 2). In contrast, pretreatment with ghrelin (100 ng/ml) decreased the apoptotic rates of CMECs (25.8±3.2% versus 38.3±1.7%, P<0.05). Regarding apoptosis, Western blot used to analysis cleaved-caspase 3 expression showed enhancement in the HCY group but it decreased in the ghrelin pretreated group (Figure 2C). These data demonstrate that ghrelin is anti-apoptotic for CMECs under these conditions.

3.3. Ghrelin increase in NO secretion

HCY (0.25 mmol/l) significantly decreased NO secretion in comparison with the control group (21.1±1.8 μmol/l versus 33.1±1.8 μmol/l, P<0.05; Figure 3A). However, pretreatment with ghrelin (100 ng/ml) enhanced the NO secretion both in basal (51.4±3.3 μmol/l versus 33.1±1.8 μmol/l, P<0.05) and in HCY-induced groups (29.9±1.6 μmol/l versus 21.1±1.8 μmol/l, P<0.05). Western blot of the expression of eNOS proteins in CMECs analysis showed that HCY down-regulated it (P<0.05), whereas pretreatment with ghrelin up-regulated expression (P<0.05). The data indicated that ghrelin pretreatment increased NO release in both basal and HCY-induced groups by increasing the expression of eNOS (Figure 3B).

3.4. Ghrelin inhibition of HCY-induced ICAM-1, IL-6, IL-8 and MCP-1 production in CMECs

To determine whether ghrelin inhibits basal and HCY-induced inflammatory response in CMECs, pro-inflammatory cytokines and adhesion molecules secretion were analysed by ELISA. The levels of ICAM-1, IL-6, IL-8 and MCP-1 in CMECs increased significantly in the HCY (0.25 mmol/l) group compared with the control group (P<0.05; Table 1). Pretreatment with ghrelin (100 ng/ml) inhibited HCY-induced ICAM-1, IL-6, IL-8 and MCP-1 release. However, ghrelin did not inhibit basal ICAM-1, IL-6, IL-8 and MCP-1 release in the absence of stimulation with HCY. Expression of IL-6 and ICAM-1 increased in the HCY-induced group compared with control group (P<0.05). After pretreatment with ghrelin, IL-6 and ICAM-1 expression was significantly decreased (P<0.05) compared with the HCY-induced group (Figures 4A and 4B).


Table 1 Ghrelin inhibited HCY-induced inflammation cytokine production in CMECs

Data shown as means±S.D. (n = 5, each group).

Cytokine Control Ghrelin HCY Ghrelin+HCY
ICAM-1 (ng/ml) 27.09±2.02 20.22±3.05 39.05±7.71* 21.09±3.20#
IL-6 (pg/ml) 58.45±7.65 35.88±8.79 119.5±7.11* 74.46±6.18#
MCP-1 (pg/ml) 62.12±3.56 54.13±4.58 89.37±6.99* 75.23±3.12#
IL-8 (pg/ml) 113.25±4.67 92.32±7.91 186.23±9.34* 152.38±4.95#


P<0.05 versus control, #P<0.05 versus HCY (0.25 mmol/l).



3.5. Ghrelin inhibition of HCY-induced NF-κB activation

NF-κB activation plays a key role in the production of chemotactic cytokines and adhesion molecule expression. HCY is a potent activator of NF-κB. Western blot assay showed that nuclear translocation of p65 protein induced by HCY was markedly attenuated by ghrelin. Ghrelin also reduced nuclear translocation of p65 protein in the absence of HCY, suggesting that the peptide inhibits NF-κB activity under basal conditions (Figures 5A and 5B).

4. Discussion

As an independent risk factor for coronary heart disease, hyperhomocysteinaemia has been the focus of many investigations, and it is an independent cardiovascular marker that could be applied in the evaluation of a patient's cardiovascular risk profile (Schnyder et al., 2002). Previous studies identified receptors for ghrelin in blood vessels and endothelial cells, suggesting that the peptide could play a modulator role in cardiovascular function (Gnanapavan et al., 2002). Our novel finding is that HCY can inhibit CMEC proliferation, induce apoptosis, and decrease NO secretion, indicating that HCY may be a risk factor in CMECs damage. In the HCY-induced cellular injury model, cell proliferation and NO secretion increased significantly, and the expression of PCNA and eNOS also increased after pretreatment with ghrelin. Additionally, pretreatment with ghrelin could inhibit apoptosis, which indicated that ghrelin may have a protective effect on HCY-induced CMECs impairment. Furthermore, we demonstrated that ghrelin inhibited NF-κB activation in CMECs, suggesting a potential mechanism for its anti-inflammatory effects.

Previous studies have suggested that lowering HCY levels could improve endothelium-dependent vasodilation, exercise performance and reduce exercise-induced myocardial ischaemia in patients with coronary heart disease and hyperhomocysteinaemia (Dinckal et al., 2003). Elevated levels of HCY may stimulate proliferation of vascular smooth muscle cells and impair endothelium function (Moat et al., 2004). A correlation was found between CMEC dysfunction and HCY treatment. On the one hand, HCY impaired CMECs proliferation and NO secretion; on the other hand, HCY induced apoptosis of CMECs. The mechanisms responsible for these deleterious effects remain to be elucidated.

Circulating levels of ghrelin- or proghrelin-derived peptides are altered in CVDs. such as cardiac ischaemia (Ozbay et al., 2008), type 2 diabetes mellitus and obesity (Huda et al., 2008), in which endothelial dysfunction plays a central role in pathology. We found that ghrelin appears to mediate the up-regulation of NO in CMECs. Previous studies showed that ghrelin increases NO production in a number of ways. Xu et al. (2008) showed that ghrelin activated eNOS in cultured endothelial cells and intact vessels. Iantorno et al. (2007) using a signalling pathway that involves PI3K (phosphoinositide 3-kinase), Akt (also known as protein kinase B) and eNOS found that ghrelin acutely stimulated production of NO in endothelium. We found that ghrelin pretreatment increased eNOS activity, thus increasing NO expression, which might be the mechanism behind its favourable effects on neovascularization and inhibition of apoptosis.

Induction of chemotactic cytokines, such as IL-6, IL-8 and MCP-1, might play an important role in monocyte recruitment and adhesion to endothelial cells in the progression of atherosclerosis (Gerszten et al., 1999). ICAM-1 was found to be up-regulated at sites prone to development of atherosclerosis (Nakashima et al., 1998). Interestingly, hyperhomocysteinaemia has been associated with elevated levels of pro-inflammatory cytokines and adhesion molecules, thought to contribute to increased cardiovascular morbidity in these patients (Wang et al., 2002; Shai et al., 2004). The mechanisms responsible for enhanced pro-inflammatory cytokine production in hyperhomocysteinaemia remain to be elucidated. Ghrelin can have a protective effect in the porcine coronary artery (Hedayati et al., 2009), and chronic subcutaneous administration of ghrelin improved LV (left ventricular) dysfunction and attenuated the development of LV remodelling and cardiac cachexia in rats with chronic heart failure (Nagaya et al., 2001). We found that HCY could increase the expression of IL-6, IL-8, MCP-1 and ICAM-1 in CMECs, while this effect could be inhibited significantly by pretreatment with ghrelin. Thus ghrelin can alleviate the impairment of HCY on CMECs. Our findings raise the possibility that administration of ghrelin in hyperhomocysteinaemia patients potentially has anti-inflammatory effects and decrease incidence of atherosclerosis.

NF-κB is a key factor in the regulation of signal transduction (Nam, 2006), which has played crucial role in inflammation, cell proliferation, differentiation and apoptosis. NF-κB is also involved in several critical functions of endothelial cells such as angiogenesis and tissue repair (Kanaji et al., 2011). A variety of stimuli including growth factors, pharmacological agents and oxidant stress coalesce on NF-κB activation. The mammalian NF-κB family consists of p65 (RelA), RelB, c-Rel, p50/p105 (NF-κB1) and p52/p100 (NF-κB2). A high concentration of HCY can activate NF-κB p65, increase the inflammatory factors generated by endothelial cells, and impair endothelial cell function, which promote atherosclerotic formation (Cheung et al., 2008). In its inactive form, NF-κB p65 is sequestered in the cytoplasm, bound by members of the IκB (inhibitory κB) family of inhibitor proteins. Phosphorylation of IκB by an IκB kinase complex exposes nuclear localization signals on the NF-κB subunits and induces translocation of the molecule to the nucleus. NF-κB binds in the nucleus, with consensus sequences of various genes, activating their transcription (Gilmore, 2006). Our observations that ghrelin attenuated HCY-induced nuclear translocation of NF-κB suggest a mechanism for the anti-inflammatory effects of ghrelin. However, the specific mechanism leading to inhibition of NF-κB activation by ghrelin needs further investigation. The PI3K/Akt signal pathway may participate in the protective effect of ghrelin (Wang et al., 2010), but other survival kinases may also play a role.

Using an in vitro cell model to determine the effect of ghrelin on HCY-induced CMECs dysfunction, we found that, although it was useful for determining the effects of ghrelin on HCY-induced CMECs, it also has some limitations. Since ghrelin peptides circulate in two distinct forms, AG (acylated ghrelin) and UAG (unacylated ghrelin), they may play different roles in the pathogenesis of specific diseases (Kojima and Kangawa, 2005; Isgaard et al., 2008). To elucidate systematically this issue, future studies should examine the role of AG and UAG on inflammatory response in CMECs.

In conclusion, HCY can inhibit proliferation and NO secretion in CMECs, while inducing apoptosis and the release of pro-inflammatory cytokines and adhesion molecules. Ghrelin pretreatment can alleviate HCY's affect, possibly through inhibition of the NF-κB signal pathway. This has important implications for at-risk patients with high HCY, where ghrelin may hold promise for prevention and treatment.

Author contribution

Dongjuan Wang, Haichang Wang, Peng Luo and Feng Cao conceived and designed the experiments. Dongjuan Wang, Haichang Wang, Peng Luo, Zheng Zhang, Nan Liu, Shenxu Wang and Yabin Wang performed the experiments. Dongjuan Wang, Peng Luo, Dongdong Sun, Zheng Zhang, Chengxiang Li and Feng Cao analysed the data. Haichang Wang, Dongdong Sun, Yabin Wang, Nan Liu and Chengxiang Li contributed reagents, materials and/or analysis tools. Dongjuan Wang, Andrew Hwang and Feng Cao wrote the paper.

Funding

This study was supported by the NSFC (National Natural Science Foundation of China) [grant numbers 81090274, 30970845, FCao 10MA024 2010CXTD01 and 2012CB518102].

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Received 14 April 2011/18 January 2012; accepted 17 February 2012

Published as Cell Biology International Immediate Publication 17 February 2012, doi:10.1042/CBI20110235


© The Author(s) Journal compilation © 2012 International Federation for Cell Biology


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)