|Year : 2016 | Volume
| Issue : 4 | Page : 644-649
Serum sex hormone and growth arrest-specific protein 6 levels in male patients with coronary heart disease
Rui Zhao, Yan Li, Wen Dai
Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
|Date of Submission||22-Jun-2015|
|Date of Decision||14-Aug-2015|
|Date of Acceptance||16-Nov-2015|
|Date of Web Publication||26-Feb-2016|
Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province
Source of Support: None, Conflict of Interest: None
Epidemiological studies have shown a high prevalence of low serum testosterone levels in men with cardiovascular disease. Moreover, the tyrosine kinase receptor Axl, the ligand of which is growth arrest-specific protein 6 (GAS6), is expressed in the vasculature, and serum GAS6 levels are associated with endothelial dysfunction and cardiovascular events. Testosterone regulates GAS6 gene transcription directly, which inhibits calcification of vascular smooth muscle cells and provides a mechanistic insight into the cardioprotective action of androgens. This study was designed to determine the correlation between serum GAS6 and testosterone levels in male patients with coronary heart disease (CHD). We recruited 225 patients with CHD and 102 apparently healthy controls. Serum concentrations of GAS6 and soluble Axl were quantified by an enzyme-linked immunosorbent assay. Levels of high-sensitivity C-reactive protein, testosterone, estradiol, and other routine biochemical markers were also measured. Testosterone decreased from 432.69 ± 14.40 to 300.76 ± 6.23 ng dl−1 (P < 0.001) and GAS6 decreased from 16.20 ± 0.31 to 12.51 ± 0.19 ng ml−1 (P < 0.001) in patients with CHD, compared with control subjects. Multiple linear regression analysis showed that serum testosterone and GAS6 levels were positively associated in male patients with CHD. Alterations in GAS6 levels may influence the development of CHD. Downregulation of GAS6/Axl signaling in the presence of low sex hormone levels during disease progression is a potential mechanism by which GAS6 affects CHD. This study provides novel results regarding the influence of sex hormones on serum GAS6 levels in patients with CHD.
Keywords: coronary heart disease; growth arrest-specific protein 6; soluble Axl; testosterone
|How to cite this article:|
Zhao R, Li Y, Dai W. Serum sex hormone and growth arrest-specific protein 6 levels in male patients with coronary heart disease. Asian J Androl 2016;18:644-9
|How to cite this URL:|
Zhao R, Li Y, Dai W. Serum sex hormone and growth arrest-specific protein 6 levels in male patients with coronary heart disease. Asian J Androl [serial online] 2016 [cited 2017 Jul 24];18:644-9. Available from: http://www.ajandrology.com/text.asp?2016/18/4/644/172825 - DOI: 10.4103/1008-682X.172825
| Introduction|| |
Coronary heart disease (CHD) is a consequence of atherosclerotic disease that causes serious damage to the coronary arteries and represents a major cause of mortality worldwide. Most epidemiological studies have found a high prevalence of low testosterone levels in men with CHD, and this association exists regardless of the patients' age.  Evidence also suggests that low testosterone levels should be considered as an independent cardiovascular risk factor. ,,
GAS6 was identified in 1988 and was further characterized in mouse embryonic NIH 3T3 fibroblasts in 1993. , GAS6 is involved in processes related to proliferation, differentiation, and inflammation in adipocytes, endothelial cells, vascular smooth muscle cells (VSMCs), and bone marrow cells and in tissues including ovary, heart, and kidney. ,, GAS6 is a secreted Vitamin K-dependent protein in serum-starved fibroblasts  and shares its domain organization and ~44% amino acid identity with the anticoagulant protein S.  GAS6 interacts with receptor tyrosine kinases of the TAM (Tyro-3, Axl, Mer) family via its C-terminal sex hormone binding globulin-like domain.  GAS6 and the soluble receptor form of Axl (sAxl) are present in mouse and human circulatory systems.  The vasculature is an important target of the GAS6/Axl system. Vascular endothelial cells, VSMCs, and fibroblasts in the vascular system synthesize and express GAS6 and Axl. , The GAS6/Axl system has been implicated in different cellular behaviors including survival and proliferation, cell adhesion and migration, homeostasis, and inflammatory cytokine release. , Studies have reported that serum concentrations of sAxl and GAS6 are elevated in patients with heart failure and acute coronary syndrome. , However, others found that plasma levels of GAS6 are decreased in patients undergoing coronary artery bypass grafting or with acute coronary syndrome. , These results indicate that GAS6 reflects the presence of common cardiovascular risk factors and can predict cardiovascular events.
Recent studies have indicated that sex hormones regulate GAS6 gene transcription, ,, suggesting a potential role of these hormones in the GAS6/Axl system. The functions of the GAS6/Axl system, and its role in inflammation and vascular diseases may be particularly relevant to CHD in humans. However, little is known about the clinical significance of the GAS6/Axl system and its association with sex hormones, particularly in male patients with CHD. We have addressed this issue by conducting a cross-sectional study to determine whether serum GAS6 levels are associated with sex hormones in male patients with CHD. In this study, we show that GAS6 and testosterone concentrations correlate to male patients with CHD. We analyzed the relationship between GAS6 and testosterone with other parameters that are altered in male patients with CHD. The results indicate that decreased testosterone may play a role in downregulating the GAS6/Axl signaling system to induce CHD.
| Materials and Methods|| |
All subjects (225 disease and 102 control cases) were recruited from the Department of Cardiology at the Renmin Hospital of Wuhan University. All cases underwent coronary angiography. Individuals found to have ≥70% occlusion of at least one major coronary artery were defined as having CHD.  Control subjects exhibited completely normal coronary arteries.
This study was approved by the Medical Ethics Review Committee of Renmin Hospital, Wuhan University. All study participants were required to provide written informed consent in accordance with policies of the Renmin Hospital of Wuhan University Ethics Committee.
Blood samples were collected from an antecubital vein from all subjects in the morning after fasting for at least 8 h. Blood samples were allowed to clot at 25°C and were later centrifuged at 25°C to obtain serum. The serum was collected, distributed into 2-ml tubes, and kept at −80°C until analysis.
A commercial enzyme-linked immunosorbent assay kit purchased from Cusabio Inc., (Wuhan, Hubei Province, China) was used to measure human GAS6 and sAxl levels in crude serum samples. All serum samples were also used to determine total bilirubin, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine, uric acid, glucose, lipid profiles, and high-sensitivity C-reactive protein (hs-CRP) by a Siemens Advia 2400 automatic biochemistry analyzer (Siemens, Erlangen, Germany). Testosterone and estradiol levels were measured by a Siemens Advia Centaur CP (Siemens, Erlangen, Germany). White blood cell counts were determined by a SysmexXN-20 (Sysmex, Kobe, Japan).
The normality of the distribution variables is expressed as means ± standard error (s.e.). Skewed data are expressed as median and interquartile ranges. Before the statistical analyses, the normality of the distribution and the homogeneity of the variables were evaluated using Levene's test. Differences in age, body mass index, GAS6, triglycerides, and HDL-C, were analyzed using independent t-tests. Other analyses used the Mann-Whitney U-test for comparisons of the quantitative variables. Correlations between GAS6 levels and biochemical variables in CHD and control groups were analyzed using Spearman's correlation analyses. Univariate and multivariate logistic regression analyses were performed to evaluate the relationship between the incidence of CHD and the measured parameters. The relationships between GAS6 and cardiovascular risk factors were tested with multiple linear regression analyses. All P values reported are two-sided, and those <0.05 were considered statistically significant. The statistical analyses were performed using IBM SPSS version 19.0 statistics software (Chicago, IL, USA).
| Results|| |
Characteristics of the study population
The biochemical variables of the control and CHD groups are summarized in [Table 1]. CHD patients had higher triglyceride, LDL-C, and white blood cell levels than did the control subjects. [Table 1] and [Figure 1] show that serum testosterone, GAS6, sAxl levels, and the testosterone/estradiol ratio, were lower in the CHD than the control group (P < 0.001 and P = 0.001, respectively). In contrast, hs-CRP levels were higher (P < 0.001) and estradiol levels were unchanged in CHD patients compared to the control subjects.
|Figure 1: Serum levels of T ( a ), E2 ( b ), T/E2 ratio ( c ), GAS6 ( d ), sAxl ( e ) and hs-CRP ( f ) in control subjects (controls) and patients with coronary heart disease (CHD). Data are presented as medians and interquartile ranges because these values were not distributed normally.|
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|Table 1: Characteristics of biochemical data among control subjects and matched CHD patients|
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Correlation between GAS6 levels and biochemical variables in CHD and control groups
Correlational analyses were performed to evaluate whether other commonly used biochemical markers correlated with GAS6 in CHD patients. In both the control and CHD groups, after adjustment for age, serum GAS6 levels were positively correlated with testosterone and sAxl levels ([Table 2] and [Figure 2]). The results showed that CHD patients with low serum GAS6 levels appeared to have lower serum testosterone levels.
|Figure 2: Scatter diagrams illustrating the correlation between serum GAS6 and sAxl ( a , b ), and the correlations between GAS6 and testosterone ( c , d ), in control subjects (controls) and patients with coronary heart disease (CHD).|
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|Table 2: Spearman partial correlation coefficients between GAS6 levels and biochemical variables in CHD and control groups|
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Logistic regression analyses for CHD
To assess the factors predicting the incidence of CHD, univariate and multivariate logistic regression analyses were performed ([Table 3]). Univariate analysis showed that glucose, LDL-C, triglycerides, testosterone, white blood cells, hs-CRP, sAxl, and GAS6 levels were associated with the incidence of CHD. Multivariate analysis showed that testosterone, GAS6, sAxl, and hs-CRP levels were predictors of the incidence of CHD.
|Table 3: Univariate and multivariate logistic regression analysis for CHD|
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Association between GAS6 and the main cardiovascular risk factors in CHD
The association between GAS6 and cardiovascular risk factors was further assessed by multivariate regression analysis. As shown in [Table 4], in male patients with CHD, GAS6 had a negative association with triglycerides and a positive association with testosterone.
|Table 4: Multiple regression analysis for GAS6 and cardiovascular risk factors|
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| Discussion|| |
To our knowledge, this is the first study examining the relationship between serum GAS6 and testosterone levels in male patients with CHD. In this study, we showed that serum GAS6 and testosterone levels were much lower in male patients with CHD than in control subjects. Moreover, serum GAS6 concentrations were positively associated with testosterone concentrations. Multiple linear regression analysis also suggested that serum levels of GAS6 and testosterone were closely associated in male patients with CHD.
Testosterone might directly activate androgen-response elements (AREs) of the GAS6 gene to influence GAS6 gene expression and protein production.  GAS6 is a potential surrogate marker of inflammation and endothelial dysfunction.  Therefore, we concluded that the involvement of GAS6 in inflammation and endothelial dysfunction was a significant factor in CHD. Our findings also indicated that decreased testosterone might play a role in downregulating the GAS6/Axl system to induce CHD. Most actions of androgen are mediated by the androgen receptor to activate transcription by binding to AREs in the GAS6 gene promoter, which inhibits VSMC calcification.  This potential mechanism can explain the upregulation of the GAS6 gene by testosterone.
Jiang et al.  have demonstrated that GAS6 levels are significantly lower in stable angina pectoris and acute coronary syndrome patients than in control patients, and have indicated a significant correlation between the degree of CVD and plasma GAS6 levels. GAS6 is derived from endothelial cells, fibroblasts, and VSMCs, and can be released into the circulation in response to disease. , GAS6 is also very important for the phagocytosis of apoptotic cells. , In dendritic cells, GAS6 can modulate the inflammatory response by downregulating TNF-α, IL-6, and interferon.  GAS6 and Axl are involved in activating the endothelium in response to inflammation, increasing the extravasation of leukocytes, and the rejection of transplanted tissues. 
GAS6 and sAxl are present in normal human serum and plasma.  Strong evidence from mouse models has suggested that the GAS6/Axl signaling pathway plays an important role in the vasculature via the PI3K/Akt pathway, resulting in cell survival, proliferation, adhesion, and protection from cellular death.  For example, GAS6 promotes the survival and migration of VSMCs, , and is overexpressed, along with its receptor Axl, in rat arterial neointima formation following balloon injury.  Jin et al. demonstrated that GAS6 plays a role in cell cycle arrest by promoting the G1/S phase transition and alleviating senescence in VSMCs. Axl plays a key role in this process, which is mediated by the binding of GAS6 thereby activating the PI3K/Akt/FoxO signaling pathway.  Axl is also involved in maintaining the integrity of the vasculature, and its expression is upregulated at sites of vascular injury, suggesting a role for Axl in vascular remodeling.  The GAS6/Axl system can mediate potential proinflammatory signals, and this may be a particularly relevant pathway for inflammatory disease conditions such as CHD. Our results demonstrated that serum GAS6 and testosterone levels were significantly lower in male patients with advanced CHD than in control subjects. Testosterone deficiency may, at least in part, be attributed to low GAS6 levels as well as reduced GAS6/Axl signaling in male CHD patients. These findings suggest that GAS6/Axl signaling is related to endothelial dysfunction and that GAS6/Axl signaling may have a crucial role in the pathogenesis of atherosclerosis through testosterone.
Testosterone is a vasoactive hormone that predominantly exerts vasodilatory effects on several vascular beds. Acute and chronic testosterone administration increases coronary artery diameter and flow, improves cardiac ischemia and symptoms in men with chronic stable angina, and reduces peripheral vascular resistance in chronic heart failure. Testosterone can also beneficially enhance biological processes involved in atheroprotection, in particular, lipid deposition and inflammation both within the arterial wall and in the circulation.  Testosterone deficiency is highly prevalent in men with CVD and is associated with an increased mortality. Low testosterone also has an adverse effect on several cardiovascular risk factors including insulin resistance, diabetes, dyslipidemia, central adiposity, and endothelial dysfunction.  Testosterone may decrease this risk, perhaps through modification and improvement of CVD risk factors, and offer potential therapeutic benefits for patients with CVD. 
The effect of testosterone replacement therapy is controversial, , although testosterone deficiency is associated with CVD in men.  Androgens act mainly through the transcriptional control of target genes mediated by the nuclear androgen receptor. , The receptor-dependent action of androgens protects against angiotensin II-induced vascular remodeling.  Some studies showed that apoptosis plays a central role in Pi-induced VSMC calcification through downregulation of the GAS6-mediated survival pathway. , Androgens prevented VSMC apoptosis and restored GAS6 expression and Akt survival signaling. These inhibitory effects of androgens on apoptosis and calcification were eliminated by flutamide and GAS6 siRNA.  A decrease in circulating testosterone levels may, therefore, lead to the downregulation of GAS6 expression, either locally in the aortic tissues or systemically in the vasculature.
The biological significance of the co-expression of GAS6 and Axl proteins in CHD patients remains to be clarified but may be related to the pathogenesis of advanced CVD in the presence of low testosterone levels. More longitudinal studies are required to elucidate the clinical significance of circulating GAS6 levels in the development of metabolic or cardiovascular diseases in human adults. One of the main limitations of this study was the small sample size resulting in the high variability of the GAS6 levels. Thus, further larger scale studies are required to confirm these results. The results of the current study are the first to show that circulating GAS6 levels are associated with CHD and that GAS6 levels could be used as a nonconventional risk factor in advanced CHD.
| Author Contributions|| |
RZ carried out the entire index test for all samples, performed the statistical analyses, and drafted the manuscript. YL conceived the study, participated in its design, and coordinated and revised the manuscript. WD collected the patient information and helped draft the manuscript. All authors read and approved the final version of the manuscript.
| Competing Interests|| |
All authors declare no competing interests.
| Acknowledgments|| |
This study was supported by the National Clinical Key Specialty Construction Projects.
| References|| |
Jones TH. Testosterone deficiency: a risk factor for cardiovascular disease? Trends Endocrinol Metab
2010; 21: 496-503.
Jones TH. Effects of testosterone on type 2 diabetes and components of the metabolic syndrome. J Diabetes
2010; 2: 146-56.
Jones TH, Saad F. The effects of testosterone on risk factors for, and the mediators of, the atherosclerotic process. Atherosclerosis
2009; 207: 318-27.
Schneider C, King RM, Philipson L. Genes specifically expressed at growth arrest of mammalian cells. Cell
1988; 54: 787-93.
Manfioletti G, Brancolini C, Avanzi G, Schneider C. The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol
1993; 13: 4976-85.
Maquoi E, Voros G, Carmeliet P, Collen D, Lijnen HR. Role of Gas-6 in adipogenesis and nutritionally induced adipose tissue development in mice. Arterioscler Thromb Vasc Biol
2005; 25: 1002-7.
Bellido-Martin L, de Frutos PG. Vitamin K-dependent actions of Gas6. Vitam Horm
2008; 78: 185-209.
Stitt TN, Conn G, Gore M, Lai C, Bruno J, et al.
The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell
1995; 80: 661-70.
Ekman C, Stenhoff J, Dahlback B. Gas6 is complexed to the soluble tyrosine kinase receptor Axl in human blood. J Thromb Haemost
2010; 8: 838-44.
Clauser S, Meilhac O, Bieche I, Raynal P, Bruneval P, et al.
Increased secretion of Gas6 by smooth muscle cells in human atherosclerotic carotid plaques. Thromb Haemost
2012; 107: 140-9.
Hurtado B, Munoz X, Recarte-Pelz P, Garcia N, Luque A, et al.
Expression of the Vitamin K-dependent proteins GAS6 and protein S and the TAM receptor tyrosine kinases in human atherosclerotic carotid plaques. Thromb Haemost
2011; 105: 873-82.
Godowski PJ, Mark MR, Chen J, Sadick MD, Raab H, et al.
Reevaluation of the roles of protein S and Gas6 as ligands for the receptor tyrosine kinase Rse/Tyro 3. Cell
1995; 82: 355-8.
Batlle M, Recarte-Pelz P, Roig E, Castel MA, Cardona M, et al.
AXL receptor tyrosine kinase is increased in patients with heart failure. Int J Cardiol
2014; 173: 402-9.
Liu YW, Yang QF, Zuo PY, Xiao CL, Chen XL, et al.
Elevated serum levels of soluble Axl in acute coronary syndrome. Am J Med Sci
2014; 349: 124-9.
Lee CH, Shieh YS, Tsai CS, Hung YJ, Tsai YT, et al.
Plasma concentrations predict aortic expression of growth-arrest-specific protein 6 in patients undergoing coronary artery bypass grafting. PLoS One
2013; 8: e79452.
Jiang L, Liu CY, Yang QF, Wang P, Zhang W. Plasma level of growth arrest-specific 6 (GAS6) protein and genetic variations in the GAS6 gene in patients with acute coronary syndrome. Am J Clin Pathol
2009; 131: 738-43.
Son BK, Akishita M, Iijima K, Ogawa S, Maemura K, et al.
Androgen receptor-dependent transactivation of growth arrest-specific gene 6 mediates inhibitory effects of testosterone on vascular calcification. J Biol Chem
2010; 285: 7537-44.
Mo R, Tony Zhu Y, Zhang Z, Rao SM, Zhu YJ. GAS6 is an estrogen-inducible gene in mammary epithelial cells. Biochem Biophysl Res Commun
2007; 353: 189-94.
Yu W, Zheng H, Lin W, Tajima A, Zhang Y, et al.
Estrogen promotes Leydig cell engulfment by macrophages in male infertility. J Clin Invest
2014; 124: 2709-21.
Gensini GG. A more meaningful scoring system for determining the severity of coronary heart disease. Am J Cardiol
1983; 51: 606.
Hung YJ, Lee CH, Chu NF, Shieh YS. Plasma protein growth arrest-specific 6 levels are associated with altered glucose tolerance, inflammation, and endothelial dysfunction. Diabetes Care
2010; 33: 1840-4.
Melaragno MG, Wuthrich DA, Poppa V, Gill D, Lindner V, et al.
Increased expression of Axl tyrosine kinase after vascular injury and regulation by G protein-coupled receptor agonists in rats. Circ Res
1998; 83: 697-704.
Nakano T, Kawamoto K, Higashino K, Arita H. Prevention of growth arrest-induced cell death of vascular smooth muscle cells by a product of growth arrest-specific gene, gas6. FEBS Lett
1996; 387: 78-80.
Hall MO, Obin MS, Heeb MJ, Burgess BL, Abrams TA. Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epithelial cells. Exp Eye Res
2005; 81: 581-91.
Ishimoto Y, Ohashi K, Mizuno K, Nakano T. Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6. J Biochem
2000; 127: 411-7.
Rothlin CV, Ghosh S, Zuniga EI, Oldstone MB, Lemke G. TAM receptors are pleiotropic inhibitors of the innate immune response. Cell
2007; 131: 1124-36.
Tjwa M, Bellido-Martin L, Lin Y, Lutgens E, Plaisance S, et al.
Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets, and leukocytes. Blood
2008; 111: 4096-105.
Hafizi S, Dahlback B. Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases. Cytokine Growth Factor Rev
2006; 17: 295-304.
Fridell YW. GAS6 Induces Axl-mediated chemotaxis of vascular smooth muscle cells. J Biol Chem
1998; 273: 7123-6.
Jin CW, Wang H, Chen YQ, Tang MX, Fan GQ, et al.
Gas6 delays senescence in vascular smooth muscle cells through the PI3K/Akt/FoxO signaling pathway. Cell Physiol Biochem
2015; 35: 1151-66.
Kelly DM, Jones TH. Testosterone: a vascular hormone in health and disease. J Endocrinol
2013; 217: R47-71.
Kelly DM, Jones TH. Testosterone and cardiovascular risk in men. Front Horm Res
2014; 43: 1-20.
Hwang K, Miner M. Controversies in testosterone replacement therapy: testosterone and cardiovascular disease. Asian J Androl
2015; 17: 187-91.
Ong PJ, Patrizi G, Chong WC, Webb CM, Hayward CS, et al.
Testosterone enhances flow-mediated brachial artery reactivity in men with coronary artery disease. Am J Cardiol
2000; 85: 269-72.
Khera M. Controversies in testosterone supplementation therapy. Asian J Androl
2015; 17: 175-6.
Laughlin GA, Barrett-Connor E, Bergstrom J. Low serum testosterone and mortality in older men. J Clin Endocrinol Metab
2008; 93: 68-75.
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, et al.
The nuclear receptor superfamily: the second decade. Cell
1995; 83: 835-9.
Heinlein CA, Chang C. Androgen receptor (AR) coregulators: an overview. Endocr Rev
2002; 23: 175-200.
Ikeda Y, Aihara K, Yoshida S, Sato T, Yagi S, et al.
Androgen-androgen receptor system protects against angiotensin II-induced vascular remodeling. Endocrinology
2009; 150: 2857-64.
Son BK, Kozaki K, Iijima K, Eto M, Kojima T, et al.
Statins protect human aortic smooth muscle cells from inorganic phosphate-induced calcification by restoring Gas6-Axl survival pathway. Circ Res
2006; 98: 1024-31.
Son BK, Kozaki K, Iijima K, Eto M, Nakano T, et al.
Gas6/Axl-PI3K/Akt pathway plays a central role in the effect of statins on inorganic phosphate-induced calcification of vascular smooth muscle cells. Eur J Pharmacol
2007; 556: 1-8.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]