Association for Academic Surgery
Low testosterone elevates interleukin family cytokines in a rodent model: a possible mechanism for the potentiation of vascular disease in androgen-deficient males

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Abstract

Background

Androgen deficiency (AD) is associated with increased risk of atherosclerosis, cardiovascular, and peripheral arterial disease. Although the biochemical and molecular mechanisms underlying this risk remain unclear, higher testosterone (TST) levels correlate to significant immunoprotective molecular and cellular responses. Our group has previously demonstrated that female sex hormones influence vascular pathogenesis via inflammatory-modulated matrix metalloproteinase (MMP) regulation. Here we investigated the role of AD and androgen replacement therapy in the modulation of these hormonally responsive pathways that could be playing a role in the development of vascular pathogenesis.

Methods

Aged orchiectomized male rats underwent TST supplementation per controlled release pellet implantation (0–150 mg). Young and aged intact groups served as controls. Serum was collected at 0–4 wk and analyzed by enzyme-linked immunosorbent assays, qualitative cytokine screening, and quantitative multiplex analyses. Human aortic smooth muscle cells were treated with 4,5α-dihydrotestosterone (DHT; 0–3000 nM) before or after interleukin 1β (IL-1β; 5 ng/mL) stimulation. Quantitative polymerase chain reaction and in-gel zymography was used to assay the effect on MMP expression and activity.

Results

Subphysiological, physiological, and supraphysiological levels of TST were achieved with 0.5, 2.5, and 35 mg TST pellet implants in vivo, respectively. Inflammatory arrays indicated that interleukin cytokines, specifically IL-2, IL-6, IL-10, IL-12, and IL-13, were elevated at subphysiological level of TST, whereas TST supplementation decreased interleukins. Supraphysiological TST resulted in a significant increase in MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in vivo. Pretreatment with IL-1β slightly increased membrane type 1-MMP (MT1-MMP) and MMP-2 expression at low to mid-level DHT exposure in vitro, although these trends were not statistically significant.

Conclusions

Here we demonstrate AD is a proinflammatory modulator and indicate that MMP-independent mechanisms may play a role downstream of AD-induced inflammatory signaling in dysfunctional vascular remodeling. Future in vivo studies will examine AD and TST supplementation in acute inflammatory response to vascular injury and in MMP-modulated vascular disease.

Introduction

Androgen deficiency (AD) is an increasingly prevalent diagnosis among today's aging male population. Androgen replacement therapy (ART) in patients with idiopathic AD has seen growing popularity with the advent of multiple exogenous replacement forms and is largely aimed at health maintenance and improving quality of life. However, we may underappreciate the benefits of ART in AD subjects regarding cardiovascular and vascular disease [1], [2], [3], [4], [5]. Likewise, we may not fully appreciate the consequences of androgen deprivation therapy routinely used in the treatment of patients with high-risk prostate cancer [6].

Regardless of whether a physiological consequence of primary testicular failure or a result of a medical or surgical intervention in the management of prostate cancer, AD has been associated with an increased risk of atherosclerosis, cardiovascular, and peripheral arterial disease [7], [8], [9], [10], [11]. Peripheral arterial disease is a major risk factor for cardiovascular and cerebral ischemic events, is often debilitating with claudication and loss of mobility, and can ultimately lead to limb loss. In spite of the vast magnitude of clinical and epidemiologic research investigating their correlation, the molecular and biochemical mechanisms underlying the role of AD in vascular disease remain unclear.

Both clinical and experimental studies have shown AD is associated with various autoimmune diseases [12], [13], [14] and that higher testosterone (TST) levels correlate to significant immunoprotective molecular and cellular responses [15]. Additionally, low TST levels have been linked to increased inflammatory markers in hypogonadal men [16]. It is well established that cytokines, growth factors, stress, and inflammation affect the regulation of matrix metalloproteinases (MMPs), enzymes largely responsible for vascular remodeling [17], [18], [19], [20]. Others and we have shown that female sex hormones influence vascular pathogenesis via inflammatory-modulated signaling and that there is a positive correlation with dysfunctional MMP regulation [21], [22], [23], [24], [25], [26]. Additionally, we have demonstrated a role for TST in the modulation of these MMP regulatory mechanisms and in the cellular processes of hyperplasia development in vitro [27]. Here we aim to identify other critical mechanisms modulated by AD that could be playing a role in the development of vascular pathogenesis. Our working hypothesis is that AD is acting as a proinflammatory modulator contributing to dysfunctional vascular remodeling.

Section snippets

Animal groups, induction of AD, and ART experimental design

Male Sprague–Dawley rats, beginning the study at the age of 8–10 mo (weighing 425–525 g) to best represent the older male population affected by the pathology of this study, were orchiectomized when obtained from Charles River Laboratories, Inc (Wilmington, MA). After 4 wk of hormone deficiency, each aged orchiectomized rat was randomly assigned to one of seven treatment groups with n = 6. Subcutaneous insertion of a 90-d slow-release water-soluble placebo pellet (Plac; 10 mg) or TST pellet

AD induction and ART dose response

To assay the efficiency of our experimental ART conditions and regime, a time interval dose–response curve was performed as described and resulting serum TST levels were measured via ELISA. Expected physiological and therapeutic TST levels were defined based on published TST levels of 0.66–5.4 ng/mL with a mean of 3.06 in normal male rats (parameters provided by the manufacturers). When compared with levels in Plac, AI, and YI controls (0.15 ± 0.07 ng/mL, ∗∗2.31 ± 0.66, and ∗∗2.72 ± 0.35,

Discussion

A variety of studies have shown that low TST levels are associated with increased risk of cardiovascular and vascular disease. Epidemiologic studies have shown an inverse relationship between TST levels and atherosclerosis, cardiovascular, and peripheral vascular disease [7], [8], [9], [10], [11]. Vascular risk factors such as obesity, hypertension, and dyslipidemia have been linked to primary AD [28], [29], [30], [31], [32]. Furthermore, low TST has been linked to increased intimal hyperplasia

Acknowledgment

The authors gratefully thank Eva Bukovska, MS, Wendy Packan, Trey Fisher, and Wes Spears for their technical assistance and Dr Joe Spengler for his support through the Spengler Research Gift Fund. This work was supported in part by funding from the Physician Medical Education Research Foundation.

Author contribution: B.M.F. contributed toward data collection, analysis and interpretation, and writing of article. D.J.H.M. contributed toward conception and design, data collection, analysis and

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