Patients with post-traumatic stress disorder (PTSD) have greater risk of developing cardiovascular disease (CVD). While chronically elevated plasma cholesterol and pro-inflammatory cytokines levels increase CVD risk, several studies have shown that cholesterol reduction does not reduce CVD risk. Acid sphingomyelinase (ASMase) activation has been implicated in both CVD and major depressive disorder. We investigated plasma pro-inflammatory cytokine levels, ASMase activity, and changes in sphingolipids in PTSD patients compared to healthy controls. Levels of interleukin 6, interleukin 10, interferon-γ and tumor necrosis factor-α were higher in PTSD patients than controls. Plasma ASMase activity and sphingosine 1-phosphate were higher in the PTSD group (1.6-fold and 2-fold, respectively; p<0.05). The results suggest that CVD risk factors in PTSD patients remain high despite cholesterol reduction.

Emerging studies strongly support that post-traumatic stress disorder (PTSD) secondary to combat exposure leads to an increased risk for early morbidity and mortality. It is becoming evident that psychological stress is associated with oxidative stress, including increased pro-inflammatory cytokines and peripheral markers of oxidative stress [1]. Various cytokines were investigated in studies related to Veterans world-wide suggesting that PTSD confers a pro-inflammatory state [2]. Recent studies have examined the complex interplay between the neuroendocrine and immunological changes in PTSD and showed that PTSD is associated with a pattern of poor physical health outcomes and a risk of illness in later life that is consistent with altered inflammatory responsiveness [3].

   Major depressive disorders have been associated with high activity of acid sphingomyelinase (ASMase), a lipid metabolizing enzyme that hydrolyses sphingomyelin (SM) into ceramide and phosphorylcholine, and can be inhibited with tricyclic antidepressants (TCA) [4]. Activation of ASMase can lead to interleukin-1 (IL-1) release from brain astrocytes [5], which in turn activates the hypothalamic-pituitary-adrenal axis and elevates plasma corticosterone levels [6]. Increased activity of ASMase has also been implicated in the pathophysiology of common diseases including cardiovascular disease (CVD) [7].

   To determine whether higher pro-inflammatory cytokine levels and increased CVD risk in PTSD could be correlated, we analyzed inflammatory markers and secretory ASMase (S-ASMase) activity in the plasma of both combat Veterans with PTSD and healthy individuals. While the Veterans taking statins displayed lower levels of total cholesterol, LDL cholesterol and non-HDL cholesterol; levels of pro-inflammatory cytokines were elevated compared to non-PTSD controls. The anti-inflammatory effects of statins have been described [8]; however, how statins influence HDL functionality and whether HDL retains pro- or anti-inflammatory properties are still obscure [9]. In addition, PTSD patients in the current study exhibited increased activity of the secreted form of ASMase (S-ASMase) in the plasma, and had elevated plasma levels of the pro-inflammatory sphingolipid sphingosine 1-phosphate (S1P). Our findings suggest that combat Veterans with PTSD, despite being on statin treatment, still exhibit high CVD risk factors in the form of elevated plasma levels of the pro-inflammatory cytokines and the sphingolipid S1P.

Participants

This pilot study was approved by the institutional review board at the Medical University of South Carolina (MUSC), and proper consent was obtained from each subject. Eight combat Veterans were diagnosed with PTSD using the Clinician Administered PTSD Scale – Diagnosis (CAPS-DX), and co-morbidity was assessed with the Mini-International Neuropsychiatric Interview [10-11]. A CAPS score of at least 50 and PTSD symptoms of at least moderate severity on the Clinical Global Impressions Scale [12] were used in the study. Blood samples were collected in Monoject™ lavender stopper collection tubes (#8881311743) (COVIDIEN, Mansfield, MA) with ethylene-diaminetetraacetic acid (EDTA) as anticoagulant. After plasma samples were separated, they were aliquoted into 0.5-ml aliquots and stored at –80ºC until processed.

   The control group consisted of five male subjects who were screened for healthy levels of conventional lipid panel (total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol, and triglycerides), glucose, C-reactive protein (CRP), complete blood count, platelet count, and comprehensive metabolic panel including liver and kidney function. A more detailed summary of the clinical profile of the control group has been previously published [13].

Plasma lipid profile analysis

Architect Immuno-chemistry analyzer (C16200, Abbot Diagnostics) was used to analyze the plasma lipid profile for the PTSD patients at the VA Clinical Laboratory at Ralph H. Johnson VA Medical Center, and for the healthy controls at the clinical laboratories of MUSC Medical University Hospital. Results were confirmed using the Cholestec LDX® system (Cholestec Corporation, Hayward, CA) in our laboratory.

Cytokine analysis

Plasma inflammatory cytokines IL-6, IL-10, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) were simultaneously measured using a human cytokine 4-plex panel and a Bioplex 200 instrument (Bio-Rad). The intra-assay variability for cytokines measured was 5-15% coefficient of variation (C.V.), whereas the inter-assay variability was between 6-11% C.V. Data presented are triplicate analysis for each sample.

Quantification of plasma sphingolipid species

Analyses of plasma sphingolipids were performed by High Performance Liquid Chromatography/ Mass Spectrometry/Mass Spectrometry (widely abbreviated as HPLC-MS/MS) at MUSC Lipidomics Shared Resource as previously described [13]. The equipment consisted of a Thermo Scientific Accela Autosampler and Quaternary Pump (Waltham, MA) coupled to a Thermo Scientific Quantum Access triple quadrupole mass spectrometer equipped with an ESI operating in multiple reaction monitoring positive ion mode.

Secretory acid sphingomyelinase activity assay

ASMase activity was assayed on duplicate samples using a slight modification of the protocol published by Jenkins et al. [14]. Briefly, S-ASMase activity was performed with 100 ml of human plasma and assayed with a reaction buffer containing 100 μm porcine brain sphingomyelin, 1×10⁵ cpm of choline-[methyl-¹⁴C] in micelles containing 0.2% Triton X-100 in sodium acetate buffer (250 mm, pH 5.0) with 0.1 mm ZnCl₂. The reaction was run for 60 min at 37°C, then terminated by the addition of 800 µl of chloroform/ methanol (2:1, v/v) followed by the addition of 0.2 ml of Milli-Q deionized water. After mixing and centrifugation at 2,000×g for 5 min, the upper (aqueous) phase was removed and used for liquid scintillation counting.

Statistical analysis

Data are presented as mean ± standard error of the mean; p<0.05 was considered statistically significant. Comparisons between lipid panels, sphingolipid profiles, and S-ASMase activities were performed using two-tailed student’s t test. Comparisons between cytokine profiles were performed using the Mann-Whitney rank sum test.

Effect of statin treatment in combat Veterans with PTSD

Information related to age, diagnoses, and whether the PTSD patient was treated for hyperlipidemia and depression at enrollment is summarized in Table 1.

Plasma lipid profiles of the PTSD patients were measured before and after enrollment and statin treatment (Table 2).

Patient	TC	HDL	TRG	LDL	Non-HDL	TC/HDL
Before Statin	207.29±14.5	42.71±3.9	190.00±31.5	126.43±16.9	164.57±15.1	5.11±0.6
After Statin	169.88±10.3	46.50±8.4	192.38±29.0	84.43±10.9	126.14±11.1	4.53±0.6
p values	0.052	0.703	0.957	0.063	0.069	0.520

While the average total cholesterol levels were over the recommended maximum of 200mg/dl before treatment, after statin use this level decreased to below 200mg/dl. Levels of LDL and non-HDL cholesterol were reduced with statin treatment.

Combat Veterans with PTSD have higher levels of plasma inflammatory cytokines and alterations in plasma sphingolipid profile

Plasma from PTSD patients had significantly increased levels of the pro-inflammatory cytokines IL-6, IL-10, IFN-γ, and TNF-α compared to healthy controls (Figure 1A). While plasma sphingolipids were generally higher in the PTSD group compared to the control group, of note was the significantly higher levels of S1P (Figure 1B). Additionally, levels of C18-ceramide and dihydro-S1P (dh-S1P) were also significantly higher in the PTSD group. Sub-clinical inflammation may have contributed to greater variability in levels of cytokines compared to sphingolipids.

Figure 1. Analysis of plasma samples from healthy volunteers (control, n=5) and Veterans with PTSD (n=8). A) Cytokine levels, B) Sphingolipid levels. Cer, ceramide; dh-S1P, dihydrosphingosine 1-phosphate; S1P, sphingosine 1-phosphate, values presented are means ± SE. Indicated p values in A derived from Mann-Whitney Rank Sum test; indicated p values in B derived from Student’s t-test. * p<0.05, ** p<0.01.

Combat Veterans with PTSD have higher plasma-associated ASMase activity

Levels of S-ASMase activity were measured in the plasma of the control and PTSD groups to determine whether there was a correlation between increased S-ASMase activity and PTSD. The mean activity of S-ASMase was significantly 60% higher (p=0.003) in the PTSD group compared to the healthy group (Figure 2). Therefore, increased S-ASMase activity in the plasma may be correlated with PTSD.

Figure 2. Analysis of secretory ASMase (S-ASMase) activity in plasma samples from Veterans with PTSD and healthy controls. Plasma (100 µl) was analyzed for S-ASMase activity as described in Materials and Methods. Each data point shown is a mean of duplicate determinations, group means are significantly different at  p=0.003

A major challenge in addressing the causal relationship between PTSD and poor health is that existing studies on PTSD have measured physical health using the traditional self-report measures of physical health, which may be influenced by psychological health. More reliable measures would involve factors that can be analyzed through laboratory tests. The pro-inflammatory markers measured in our study might be used to provide a diagnosis/prognosis of PTSD and concomitant diseases such as cardiovascular, gastrointestinal, and musculoskeletal disorders.

   Despite widespread use of statins to reduce CVD risk, there is considerable variability in the cholesterol-lowering response among individuals [15, 16, 17]. The origin of this variability is poorly understood; however, pharmacogenomic studies have begun to uncover associations of candidate genes with alterations in cholesterol-lowering response to statins [18, 19, 20] It appears that the genetic contribution to the variability in statin-mediated cholesterol reductions result from combined effects from multiple polymorphisms with minimal contribution from individual genes [20, 21]. Using a targeted lipidomics platform, Kaddurah-Daouk et al found no overlap of lipid changes that correlate with LDL cholesterol or CRP responses to simvastatin, which suggested that distinct metabolic pathways regulate statin effects on the two biomarkers [22]. The sphingolipidomics approach described in this study could in future large population studies provide insights into responses to statins including inflammation-related pathways.

   Despite the apparent trend of statins in lowering blood cholesterol observed in the PTSD group, pro-inflammatory cytokines and S1P were still higher than in the control group, suggesting other CVD risks such as chronic inflammation remains. A pooled analysis of four studies measuring carotid intimal-media thickness (cIMT) in randomized familial hypercholesterolemia patients concluded that while the rate of cIMT development slows considerably in response to intensive statin therapy, the complications of atherosclerosis also depend on the degree of inflammation and other physical properties of the artery [23]. Inflammatory markers such as CRP and soluble CD40 ligand have been shown to decrease in response to aggressive statin therapy [24, 25]; however, studies on patients taking various lipid-lowering drugs have shown that there remains a ‘residual risk’ in the development of CVD, perhaps because underlying inflammatory causes were not addressed [23, 26, 27, 28]. Therefore, it would appear that with statin use the duration of treatment, the type of statin, and the patients studied are influential in determining the outcome on CVD risk. Our data imply that the PTSD group in this study may still be at higher risk for development of CVD despite the statin treatment.

   Numerous studies addressed the role of cholesterol in the regulation of synaptic transmission; however, the cellular mechanisms by which cholesterol deficiency mediates the pathological events in the nervous system are poorly understood. It has been suggested that the base line of the extracellular glutamate concentration in the brain can have a major effect on the neuronal excitability and synaptic transmission. Recently, it was demonstrated that reduced levels of cholesterol in the plasma membrane of neurons causes increase in ambient glutamate [29]. In case of PTSD the application of statins may therefore exacerbate the clinical course of depression.

   Atherosclerotic plaque buildup in the vessel wall is typically initiated by activated macrophages internalizing modified LDL particles. Activated macrophages release cytokines, such as TNF-α, which can cause local inflammation and recruit more macrophages to the area. We have previously reported that S1P is able to induce increases in released TNF-α, and prostaglandin E2 in macrophages in vitro [30]. We have also reported that sphingosine kinase, the enzyme that generates S1P, is released by human monocytic cells in response to modified LDL immune complexes, generating extracellular S1P that may be involved in sustained activation [30, 31]. Our current data suggest that PTSD can lead to increased pro-inflammatory cytokines and S1P in the plasma that may drive the pro-inflammatory mechanisms contributing to the development of vascular disease; however, the significance of elevated dh-S1P levels in PTSD patients is not clear.

   It has been shown that S-ASMase can hydrolyze SM in LDL particles causing an increase in particle size and a tendency to aggregate and stick to the vessel wall [32]. This finding suggests that the doses of ASMase-inhibiting effects of TCA drugs such as Norpramin (desipramine) [4] could be optimized to further reduce plasma S-ASMase activity to non-PTSD levels. Furthermore, it has been shown that the hydrolysis of SM to ceramide by ASMases is a necessary step in the pathway for the production of S1P [33]. Our current data show that plasma ceramide levels were elevated in the PTSD patients, which support the notion that this ceramide pool can be converted to S1P.

   The relatively low number of patients merit that caution must be used in interpreting the results. For instance, despite lower levels of plasma total and LDL cholesterol levels in PTSD patients after statin treatment, statistically significant differences before and after statin treatment were not attained. The expansion of this study to include more patients would add more statistical power. In spite of the small sample size, the results of this pilot study brings up the interesting possibility of whether chronic inflammatory processes are causally involved in major depressive disorders and it is intriguing to hypothesize that lowering the pro-inflammatory symptoms may also contribute to the treatment of major depressive disorders such as PTSD.

This work was supported by NIH Grants HL-079274 and HL-079274-04S1 (ARRA), The Southeastern Clinical and Translational Research Institute (SCTR, formerly GCRC) and the South Carolina Center of Biomedical Research Excellence (COBRE) in Lipidomics and Pathobiology (P20 RR17677) to S.M.H; and funding from the Medical Research Service, Ralph H. Johnson VAMC, Charleston, to M.B.H. Special thanks to the Lipidomics Shared Resource facility at MUSC for sphingolipid analysis, and for the MUSC Proteogenomics Facility  for the use of the Bioplex system for cytokine determination.

MBH has shares in Merck, and has been awarded grants from Pfizer Inc. and Otsuka Pharmaceuticals.

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PTSD
post-traumatic stress disorder
sphingolipidomics
HPLC-MS/MS
sphingolipids
sphingomyelinase
sphingosine 1-phosphate
ceramide
cytokine
interleukin
IL-6
IL-10
TNF
tumor necrosis factor
interferon
HDL
LDL
cholesterol
hyperlipidemia
statins