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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 769-776 |
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doi:10.1111/j.1745-7270.2008.00457.x |
Inhibition of proliferation and apoptosis
of vascular smooth muscle cells by ghrelin
Min Zhang, Fang Yuan, Hua Liu, Hui Chen, Xingbiao Qiu, and Weiyi Fang*
The Affiliated Chest Hospital, Shanghai
Jiaotong University, Shanghai 200030, China
Received: April 4,
2008�������
Accepted: June 24,
2008
This work was
supported by a grant from Science and Technology Commission of Shanghai
Municipality (No. 074119635)
*Corresponding
author: Tel, 86-21-62821990; Fax, 86-21-52306186; E-mail, [email protected]
To evaluate the possible role of ghrelin in
the development of atherosclerosis, its effects on tumor necrosis factor (TNF)-a-induced proliferation and apoptosis of
vascular smooth muscle cells (VSMCs) were investigated. Rat VSMCs were
pretreated with different concentrations of ghrelin and then with TNF-a. VSMC proliferation was measured by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay� and flow
cytometry method. Apoptosis was detected using propidium iodide and Annexin-V
labeling method. Exogenous� ghrelin (10-1000 ng/ml) significantly inhibited TNF-a-induced� proliferation of VSMCs in a
concentration-dependent� manner. Treatment with 1000 ng/ml ghrelin was most
effective� at inhibiting VSMC proliferation rate and the expression of
proliferating cell nuclear antigen. However, treatment with des-acyl ghrelin
affected neither proliferation nor PCNA expression. In contrast, TNF-a-induced apoptosis of VSMCs was inhibited
by both ghrelin and des-acyl� ghrelin in concentration-dependent manners, with
maximal� �inhibition observed for both compounds at 1000 ng/ml. Taken together,
our results� suggested that ghrelin inhibited both the proliferation� and
apoptosis of rat VSMCs. Furthermore, the former effect is probably mediated by
the growth hormone secretagogue receptor type 1a receptor, while the latter
effect may be mediated� through other receptors.
Keywords������� ghrelin; vascular smooth muscle cell; atherosclerosis; proliferation; apoptosis
Ghrelin, a brain-gut peptide, was discovered in 1999. To date, it is
the only natural ligand identified for growth hormone� secretagogue receptor
(GHSR) [1]. Binding to its receptor, ghrelin is able to promote the release of
growth hormone, which is involved in the regulation of energy metabolism.
Abnormal ghrelin signaling has been implicated� in various diseases and
physiological processes. Ghrelin is abundantly distributed in the
cardiovascular system. A recent� study demonstrated that ghrelin receptor
density was higher in the cardiovascular system of athero�sclerotic mice than
in that of normal control mice, thereby suggesting� ghrelin and its receptor
might play a vital role in this disease [2]. Other studies have reported that
ghrelin exhibited� protective effects against the development of
atherosclerosis� by increasing coronary blood flow [3], improving� endothelial
function [4], inhibiting endothelial injury [5], producing vasodilation and
enhancing cholesterol� efflux in macrophages [6]. Furthermore, ghrelin has been
reported to exert anti-inflammatory effects on the cardiovascular system. For
example, ghrelin was shown to inhibit� both tumor necrosis factor (TNF)-a-induced
cytokine release and NF-kB activity in human umbilical vein endothelial� cells (HUVECs) [7].
Furthermore, we found that ghrelin down-regulated CD40 expression in HUVECs
[8], suggesting that it might be involved in regulating the inflammatory
process of atherosclerosis. Therefore, ghrelin and its receptor appear� to have
very important functions in the development of atherosclerosis.
The proliferation and apoptosis of smooth muscle cells (SMCs) are closely related to generation, development, plaque stability, postangioplasty restenosis and vein graft disease [9,10]. Multiple bioactive substances, such as TNF-a, can induce phenotypic changes in SMCs, resulting in their activation, proliferation and migration, followed by the formation of a patch fibrous cap [11]. Many cellular factors� are capable of promoting SMC apoptosis, which ultimately results in the transformation of stable plaques to unstable plaques [12]. It has not yet been reported whether ghrelin has the ability to defer the progress of atherosclerosis by inhibiting the proliferation and apoptosis of SMCs.
In the present study, cultured VSMCs were treated with exogenous ghrelin to determine its effects on VSMC proliferation� and apoptosis and to further elucidate the role of ghrelin in the development of atherosclerosis.
Materials and Methods
Materials
Wister rats, 6-7 weeks old, 200�10 g, were purchased from the Animal Center of
Shanghai Institutes for Biological� Science, Chinese Academy of Sciences
(Shanghai, China). Dulbecco�s modified Eagle�s medium was obtained from Gibco
(Gaithersburg, USA). Ghrelin and des-acyl ghrelin were obtained from Phoenix
Pharmaceuticals (Burlingame, USA). Recombinant human TNF-a,
fluorescence-activated cell sorter (FACS) permeabilizing solution and an
Annexin V-FITC apoptosis kit were purchased from BD Biosciences (San Jose,
USA). The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
was obtained from Roche (Indianapolis, USA). Dimethyl sulfoxide (DMSO) and
propidium iodide (PI) were purchased from Sigma Chemical Company (St. Louis,
USA). Anti-proliferating cell nuclear antigen (PCNA) rat monoclonal antibodies,
PC10 and immunoglobulin G (IgG) 2a, were purchased from Santa Cruz
Biotechnology� (Santa Cruz, USA). Flourescein-isothiocyanate-conjugated rabbit
anti-rat IgG was obtained from DAKO (Tokyo, Japan).
Culture and treatment of rat thoracic aortic SMCs
Thoracic aortas (100-150 g) were obtained from Wistar rats and placed in Hanks solution. The intima and extima were carefully removed. The medial smooth muscle tissues� were cut into 1 mm3 pieces and distributed equally into Carrel�s flasks. Cells from the tissues were cultured in an incubator at 37 �C under 5% CO2, followed by the addition� �of Dulbecco�s modified Eagle�s medium supplemented with 20% fetal calf serum and 100 U/ml penicillin. Cells reached confluence after 2-3 weeks and were then digested� with 0.125% trypsin, passaged and cultured continuously� [13]. The presence of VSMCs was confirmed� by light microscopy, electron microscopy and immunohistochemical analysis. Cells of the 3�5 generations �were used in assays.
After the replacement of the complete medium with fetal� bovine serum-free RPMI 1640 medium and an overnight� culture at 37 �C under 5% CO2, VSMCs were treated with different concentrations of TNF-a (1-1000 U/ml) in phosphate�-buffered saline (PBS) for varying amounts of time (1, 3, 5, 7, 9 and 12 h). An equal amount of PBS was used in negative control experiments.
After substituting the complete medium with fetal bovine� serum-free RPMI 1640 medium, VSMCs were plated in 6-well plates and pretreated with PBS (negative control), exogenous ghrelin (10, 100 or 1000 ng/ml) or des-acyl ghrelin (1000 ng/ml) for 1 h prior to a 7 h treatment with 100 U/ml TNF-a at 37 �C under 5% CO2.
Assessment of VSMC proliferation by MTT assay
MTT assays were carried out as previously described [14]. Briefly, VSMCs obtained from rat aortas were cultured in 96-well plates in 100 ml medium at 37 �C under 5% CO2 for 24-96 h, followed by a 4 h incubation with 10 ml MTT (0.5 mg/ml). Next, solubilization solution (100 ml DMSO) was added to each well and incubated overnight. Once the purple crystals had completely dissolved, the absorbance of the samples was measured at 570 nm using� a UV/VIS spectrophotometer (u-2000; Hitachi, Tokyo, Japan). Proliferation rate was calculated as following.
Eq.
Samples before intervention were used as blank controls.
Detection of PCNA expression by indirect immuno�fluorescence flow
cytometry (FCM)
VSMCs were collected from 6-well plates, placed in Eppendorf tubes, washed with PBS, fixed with 1% phosphonoformate for 30 min, washed again with PBS and centrifuged at 161 g for 10 min. The cells were then permeabilized with FACS permeabilizing solution for 10 min, washed with PBS and centrifuged at 161 g for 10 min. Cells were incubated with 50 ml IgG2a for 5 h at 4 �C and washed three times with PBS. Then, 10 ml flourescein-isothiocyanate-conjugated IgG was added to each tube for 30 min at 4 �C. After the removal of unbound� antibodies by washing, the cells were resuspended in 200 ml PBS, fixed with 1% phosphonoformate and assessed by FCM. A total of 1�104 cells were collected for analysis using FACScan (Becton Dickson, Franklin Lakes, USA) and Cell Quest software (San Jose, USA), and the mean fluorescence intensity (MFI) was calculated.
Analysis of VSMC DNA contents by PI staining
Apoptosis of VSMCs was analyzed by Annexin V staining and detected as previously reported [15]. The DNA contents� of VSMCs were determined by PI staining and FACS analysis. Attached cultured VSMCs were trypsinized, mixed with suspended cells, centrifuged at 252 g for 5 min, washed with PBS, fixed with formaldehyde, incubated with RNase and stained with PI at a final concentration� of 25 mg/ml. FCM Cell Fit 2.02 software (Microscience, Phoenix Technology, Washington, USA) was used to analyze� the stained cells.
VSMC apoptosis detected by Annexin V staining
Cultured rat aortic VSMCs were collected, washed with PBS and resuspended in 1�binding buffer to a final density� of 1�106 cells/ml. Then 100 ml aliquots of the cell suspension� were transferred into 5 ml tubes, followed by the addition of 5 ml Annexin V-FITC and 5 ml PI to each tube. The cells and reagents were thoroughly mixed and incubated at 25 �C in the dark for 15 min, and then 40 ml 1�binding buffer was added. FCM was carried out within 1 h, and suitable negative and positive controls were included. The percentages of Annexin V-positive cells were calculated and analyzed using Cell Quest software [15].
Statistical analysis
Each experiment was repeated three times. SAS software (SAS Institute, Cary, USA) was used for statistical analysis� and data were expressed as mean�SD. Comparisons between� data were carried out by single-factor analysis of variance, and differences were considered statistically significant� at P<0.05.
Results
Identified VSMCs using microscope and immuno��histo�chemistry
VSMCs were identified using light microscopy, electron microscopy and immunohistochemical analysis. Under light microscopy (Olympus, Tokyo, Japan), cells appeared spindle-shaped and were arranged into bundles. Multiple layers of cells were observed in several areas, while single layers grew in other areas. The characteristic peak-and-valley features of VSMC colonies were also evident [Fig. 1(A)]. Under electron microscopy (H-550; Hitachi), muscle fibrils parallel to the long axis and associated dense bodies were evident in the cytoplasm of the cultured VSMCs [Fig. 1(B)]. Finally, immunohistochemical analysis� using a VSMC-specific antibody revealed that over 98% of the cells were positively stained (20 fields were randomly chosen, and 100 cells in each field were evaluated) [Fig. 1(C)]. Taken together, these three tests served to confirm the identity and purity of the cultured VSMCs obtained from Wistar rats.
Effects of TNF-a on VSMC proliferation evaluated by MTT assay
VSMC proliferation was enhanced following TNF-a stimulation. The peak proliferation was found at 7 h after TNF-a treatment. By 9 h after TNF-a treatment, VSMC proliferation had decreased, and the cells began to shrink, rounded up and became detached. Stimulation of VSMCs with TNF-a at various concentrations (1-1000 U/ml) led to a concentration-dependent increase in VSMC proliferation, with the maximum proliferative response at 100 U/ml. Although� cell proliferation was markedly enhanced� as the TNF-a concentration increased, the cytotoxicity� was also enhanced, leading to increased numbers� of dead cells (Fig. 2).
Effects of ghrelin on TNF-a-induced VSMC proliferation
evaluated by MTT assay
Ghrelin (10-1000 ng/ml) significantly inhibited 100 U/ml TNF-a-induced VSMC proliferation in a dose-dependent manner. Treatment with 1000 ng/ml ghrelin decreased 100 U/ml TNF-a-induced VSMC proliferation from 0.78%�0.08% to 0.39%�0.10% (P<0.01). In contrast, treatment with 1000 ng/ml des-acyl ghrelin had no effect on 100 U/ml TNF-a-induced VSMC proliferation (0.75�0.05%; P>0.05) (Fig. 3).
Effects of ghrelin on TNF-a-induced PCNA expression�
evaluated by FCM
Exogenous ghrelin inhibited TNF-a-induced PCNA expression� in a dose-dependent manner. Treatment with 1000 ng/ml ghrelin caused the maximum inhibition, and the MFI of PCNA decreased from 782.13�66.42 to 348.35�71.56 (P<0.01). In contrast, treatment with 1000 ng/ml des-acyl ghrelin did not inhibit TNF-a-induced� PCNA expression (MFI=817.31�105.19; P>0.05) (Fig. 4).
Effects of ghrelin on TNF-a-induced apoptosis
evaluated� by PI staining
Similar dose-dependent increases in the number and proliferation� of VSMCs were obtained at TNF-a concentrations� of 10-100 U/ml. However, treatment with 1000 U/ml TNF-a caused massive decreases in cell number� and proliferation. Sub-G1 peaks were observed, suggesting extensive apoptosis of the VSMCs. Exogenous ghrelin significantly inhibited TNF-a-induced VSMC apoptosis in a dose-dependent manner. The proportion of apoptotic cells decreased by 33.19% after treatment with 1000 ng/ml ghrelin. Similarly, treatment with 1000 ng/ml des-acyl ghrelin significantly inhibited TNF-a-induced VSMC apoptosis, with the proportion of apoptotic cells decreasing by 32.50% after treatment (Fig. 5).
Effects of ghrelin on TNF-a-induced apoptosis
evaluated� by Annexin V staining
Exogenous ghrelin significantly inhibited TNF-a-induced VSMC apoptosis in a dose-dependent manner. The proportion� of apoptotic cells decreased by 27.20% after treatment with 1000 ng/ml ghrelin. Similarly, the proportion� of apoptotic cells decreased by 26.22% in response� to treatment with 1000 ng/ml des-acyl ghrelin (Fig. 6).
Discussion
VSMC proliferation and apoptosis play critical roles in the process of aortic stenosis development, patch stability and restenosis following coronary interventions. In the present study, cultured rat VSMCs were selected for experiments. MTT assays and PCNA expression levels were used to evaluate VSMC proliferation [16]. TNF-a was added to the cell cultures to simulate the activation of VSMCs and determine the effects of TNF-a on VSMC proliferation. VSMC proliferation was found to occur, even in the absence� of external stimulation by TNF-a. Nonetheless, TNF-a promoted VSMC proliferation. However, at extremely high doses or after prolonged treatment periods, TNF-a caused the cells to shrink, round up and become detached. Massive� cell death at high concentrations and after long incubation periods was noted.
The present study provided novel evidence regarding the close relationship between exogenous ghrelin and VSMC proliferation and apoptosis. Proliferation of VSMC is not only important during the development of an aortic stenosis patch, but it is also a pivotal cause of postoperative restenosis. Our results revealed that ghrelin significantly inhibited TNF-a-induced VSMC proliferation in a dose-dependent manner, suggesting that ghrelin may function by inhibiting the development of aortic stenosis. It has been reported that ghrelin, an acylated peptide with numerous activities in various organ systems, exerts most of its known effects on the body through a highly conserved G-protein-coupled receptor, GHSR type 1a (GHSR-1a) [17]. Also, there are other forms of ghrelin peptides, including des-acyl ghrelin, which lacks the acyl modification, and minor acylated ghrelin species, such as n-decanoyl ghrelin. Des-acyl ghrelin is an isomer of ghrelin that is abundantly present in serum but unable to unite with the GHSR-1a receptor. Therefore, it has none of the endocrine and inflammatory effects of ghrelin [18,19]. We found that ghrelin inhibited TNF-a-induced VSMC proliferation, whereas des-acyl ghrelin (which is incapable of binding to GHSR-1a) had no effect on VSMC proliferation. It is therefore possible that the inhibitory effects of ghrelin on VSMC proliferation are dependent on GHSR-1a. Indeed, it has been reported that exogenous ghrelin inhibits the TNF-a-induced expression of cell adhesion molecules and activation of NF-kB in HUVECs, and that these anti-inflammatory effects of ghrelin are mediated by the GHSR-1a receptor [7]. Moreover, we previously demonstrated that exogenous ghrelin significantly inhibits cytokine-induced CD40 expression in HUVECs, and that this effect of ghrelin is also mediated by GHSR-1a [8]. Taken together, these results support the supposition that the inhibitory effects of ghrelin on VSMC proliferation, its anti-inflammatory functions and its inhibitory effects on CD40 expression are all mediated by GHSR-1a.
VSMC apoptosis is involved in the generation and development of aortic stenosis and ultimately results in patch instability. In this study, PI staining and Annexin V staining were performed to analyze the cell cycle and degree of apoptosis in VSMCs. Both ghrelin and des-acyl ghrelin inhibited TNF-a-induced VSMC apoptosis, suggesting an identical GHSR-1a-independent anti-apoptotic mechanism for both ghrelin and des-acyl ghrelin. However, the observed anti-apoptotic function may be mediated by other as yet unknown receptors. For example, Baldanzi et al discovered that ghrelin and des-acyl ghrelin both inhibit apoptosis of endothelial cells via extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3 kinase/protein kinase B signaling pathways, which are independent of GSHR-1a [20]. Their results identified additional pathways, but are compatible with our present findings. In addition, Muccioli et al reported that the anti-lipolytic effects of ghrelin are not mediated through GSHR-1a [21]. Accordingly, ghrelin appears to have both anti-proliferative and anti-apoptotic effects on VSMCs, and may activate different signal transduction pathways via binding to different receptors that remain to be identified.
Although the clinical role of ghrelin in the development of coronary artery disease requires further research, its regulation of VSMC proliferation and apoptosis may have significant theoretical and clinically applicable value in the development of coronary aortic stenosis patches, patch stability and postoperative restenosis. In the course of coronary aortic stenosis and restenosis, inflammatory mediators, including cytokines, can either promote VSMC proliferation or induce apoptosis by modulating intracellular inflammatory responses. Ghrelin may inhibit VSMC proliferation and apoptosis by binding to different receptors to activate diverse signaling pathways that can ultimately defer or hinder the progress of coronary aortic stenosis and prevent restenosis.
Taken together, the results of the present study demonstrate that ghrelin mediates GHSR-1a-dependent inhibition of VSMC proliferation and GHSR-1a-independent inhibition of VSMC apoptosis. These effects of ghrelin on VSMCs may represent one mechanism by which ghrelin interferes with the development of aortic stenosis. Although this pilot study only examined the influence of ghrelin on VSMC proliferation and apoptosis in cell culture settings, it lays a solid foundation for future studies. For example, the specific parts of the signaling pathways involved in VSMC proliferation and apoptosis that are affected by ghrelin need to be addressed and characterized. Further research in this field is clearly warranted.
Acknowledgements
The authors thank Drs. Guoping Lu and Chunfang Wu (Department of Cardiology, Shanghai Ruijin Hospital, Shanghai, China) for their generous help and skillful assistance.
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