A decade of letrozole: FACE

Third-generation nonsteroidal aromatase inhibitors (AIs), letrozole and anastrozole, are superior to tamoxifen as initial therapy for early breast cancer but have not been directly compared in a head-to-head adjuvant trial. Cumulative evidence suggests that AIs are not equivalent in terms of potency of estrogen suppression and that there may be differences in clinical efficacy. Thus, with no data from head-to-head comparisons of the AIs as adjuvant therapy yet available, the question of whether there are efficacy differences between the AIs remains. To help answer this question, the Femara versus Anastrozole Clinical Evaluation (FACE) is a phase IIIb open-label, randomized, multicenter trial designed to test whether letrozole or anastrozole has superior efficacy as adjuvant treatment of postmenopausal women with hormone receptor (HR)- and lymph node-positive breast cancer. Eligible patients (target accrual, N = 4,000) are randomized to receive either letrozole 2.5 mg or anastrozole 1 mg daily for up to 5 years. The primary objective is to compare disease-free survival at 5 years. Secondary end points include safety, overall survival, time to distant metastases, and time to contralateral breast cancer. The FACE trial will determine whether or not letrozole offers a greater clinical benefit to postmenopausal women with HR+ early breast cancer at increased risk of early recurrence compared with anastrozole.
Introduction
The aromatase inhibitors (AIs) have proven to be a powerful drug class for use in hormone-sensitive breast cancer and have shown superiority over the selective estrogen-receptor modulator (SERM) tamoxifen in preclinical models of hormone-dependent breast cancer [1] and in randomized controlled trials in patients with advanced breast cancer [2] and early breast cancer [3, 4]. Initial adjuvant therapy with either letrozole (Femara®) or anastrozole (Arimidex®) was shown to be significantly more effective than tamoxifen in both the Breast International Group (BIG) 1-98 and Anastrozole and Tamoxifen Alone or in Combination (ATAC) randomized controlled trials in postmenopausal women with localized breast cancer [3, 4].
In the BIG 1-98 primary core analysis, patients with hormone receptor-positive (HR+) tumors randomized to receive letrozole initially were compared with those assigned to receive tamoxifen initially (N = 8,010). After a median follow-up of 25.8 months, 351 events had occurred in the letrozole group (n = 4,003) and 428 events in the tamoxifen group (n = 4,007), with 5–year disease-free survival (DFS) estimates of 84.0% and 81.4%, respectively. Letrozole significantly reduced the risk of breast cancer recurrence (hazard ratio = 0.81; 95% confidence interval [CI] 0.70, 0.93; P = 0.003), especially the risk of distant recurrence (hazard ratio = 0.73; 95% CI 0.60, 0.88; P = 0.001) [3]. An analysis limited to patients randomized to either letrozole-only or tamoxifen-only arms (N = 4,922) was recently published and allows for more direct comparisons with results from other trials of continuous therapy with a single endocrine agent [5]. Results from the letrozole-only or tamoxifen-only arms were consistent with those published for the primary core analysis and showed that letrozole significantly reduced the risk of DFS events (hazard ratio = 0.82; 95% CI 0.71, 0.95; P = 0.007) [5, 6] and the risk of distant metastases. After a median follow-up of 51 months, 352 DFS events (14.3%) were observed in the letrozole-only group (n = 2,463), compared with 418 DFS events (16.9%) in the tamoxifen-only group (n = 2,459) [5, 6].
The ATAC trial, which compared anastrozole with tamoxifen for 5 years, did not have receptor positivity as a study requirement. After a median follow-up of 68 months (n = 6,186), anastrozole significantly prolonged DFS (575 events with anastrozole vs. 651 events with tamoxifen; hazard ratio = 0.87, 95% CI 0.78, 0.97; P = 0.01) and time-to-recurrence (402 vs. 498 events; hazard ratio = 0.79, 95% CI 0.70, 0.90; P = 0.0005), and significantly reduced the risk of developing distant metastases (324 vs. 375 events; hazard ratio = 0.86, CI 0.74, 0.99; P = 0.04) and contralateral breast cancers (35 vs. 59 events; 42% reduction, 95% CI 12, 62; P = 0.01) in the intent-to-treat (ITT) population [4]. However, neither the time to distant recurrence nor distant DFS (DDFS) were significantly improved with anastrozole in the HR+ population [7].
While both letrozole and anastrozole have been evaluated extensively in early breast cancer, no head-to-head trial of these two AIs has been conducted in this setting. This report will focus on the design of the Femara versus Anastrozole Clinical Evaluation (FACE) trial, and describe how it will prospectively address potential efficacy and safety differences between the two AIs.
Rationale for head-to-head trial
An American Society of Clinical Oncology technology assessment concluded that AIs should be included in the adjuvant treatment of postmenopausal women with HR+ breast cancer [8] but did not recommend one AI over another. The National Comprehensive Cancer Network has also recommended initial adjuvant therapy with an AI (specifically letrozole or anastrozole) as an alternative to tamoxifen [9]. Some evidence suggests that AIs may not be equivalent, even though they belong to the same pharmacologic class of agents; differences have been reported in terms of potency, suppression of aromatization, antitumor effects, and pharmacogenomics. However, whether or not one AI is superior in treating early breast cancer is not known.
Relative potency
Bhatnagar and colleagues compared the aromatase-inhibiting potency of letrozole and anastrozole in a variety of aromatase-containing cellular endocrine and tumor models [10, 11]. While letrozole and anastrozole were approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole was found to be more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells (50% inhibitory concentration [IC50] 20 vs. 600 nM, respectively), normal human adipose fibroblasts (0.8 vs. 14 nM), and human cancer cell lines (MCF-7Ca 0.07 vs. 0.82 nM and JEG-3 0.07 vs. 0.99 nM). Miller and colleagues reported that letrozole and anastrozole were more potent than aminoglutethimide in vitro against tumor samples obtained from postmenopausal women with breast cancer, with letrozole demonstrating the lowest IC50 (2 nM, 8 nM, and 20 μM, respectively) [12]. Letrozole was compared with anastrozole in vivo in athymic mice inoculated with MCF7 cells [13]. Tumor volumes increased to 145.9% in controls and decreased to 22.4% with letrozole 10 μg, and to 95.6% or 78.2% with anastrozole 10 or 60 μg, respectively. These results are consistent with a higher in vitro potency of letrozole in cell-based assays [13].
Suppression of aromatization
The effects of letrozole and anastrozole on suppression of total-body aromatization and plasma estrogen concentrations have been compared in patients with metastatic breast cancer [14, 15]. Levels of aromatase were detectable in 11 of 12 patients during treatment with anastrozole (mean percentage inhibition in the whole group, 97.3%) but in none of the 12 patients during treatment with letrozole (> 99.1% suppression in all patients; Wilcoxon, P = 0.0022, comparing the two drug regimens). Suppression of estrone and estrone sulfate was found to be significantly greater during treatment with letrozole compared with anastrozole (P = 0.019 and P = 0.0037, respectively). Another study conducted in 54 postmenopausal women with invasive breast cancer showed that more complete inhibition of aromatase was achieved by 2.5 mg of letrozole than 1 mg of anastrozole, resulting in significantly greater suppression of estradiol (P < 0.0001) [15]. Thus, letrozole reduces estradiol levels to a greater degree than anastrozole, but it is not known whether this difference is clinically relevant.
Breast cancer proliferation
Biological changes in breast tumors occurring within 14 days of starting treatment may predict the efficacy of different endocrine agents in the adjuvant setting and could prove to be useful surrogate markers to compare drug efficacy [16]. A study of neoadjuvant endocrine therapy compared the effects of letrozole and anastrozole on the expression of HRs and markers of tumor proliferation in postmenopausal women with estrogen receptor (ER)-positive breast cancer [16]. Neoadjuvant letrozole and anastrozole decreased overall ER expression (Allred score) after 14 days, but more cases showed a reduction in progesterone receptor (PgR) expression following letrozole treatment (75/106) than with anastrozole treatment (65/102). Furthermore, only letrozole significantly reduced proliferation at lower Allred ER expression levels (scores 2–5). This is a potentially important finding, because it has been suggested that a greater suppression in proliferation may lead to improved DFS [17].
Clinical activity
Letrozole and anastrozole have not been directly compared in the adjuvant setting, but data from a randomized, head-to-head trial in patients with advanced breast cancer are available [18]. Postmenopausal women with advanced breast cancer (N = 713) that had progressed either during antiestrogen therapy or within 12 months of completing that therapy were randomized to receive letrozole (2.5 mg per day) or anastrozole (1 mg per day). Letrozole was significantly superior to anastrozole in terms of overall response rate (19.1% vs. 12.3%, P = 0.013), but there were no significant differences in median time to progression, the primary end point of the trial. Both agents were well-tolerated, and there were no significant differences in safety.
Anastrozole and letrozole in the adjuvant setting have demonstrated superiority to tamoxifen in significantly reducing the risk of recurrence. In the ATAC trial, at 68 months’ median follow-up, anastrozole significantly reduced the risk of distant metastases in the ITT population by 14% (P = 0.04) but not significantly in the HR+ patient subgroup (hazard ratio = 0.84; P = 0.06) [4]. A recent study of the recurrence rates after 2.5 and 5 years from the ATAC study showed that there were fewer recurrence events with anastrozole at these time points due to reductions in contralateral, primary, loco-regional, and distant recurrences [19]. In addition, at 25.8 months’ median follow-up in the BIG 1-98 trial, letrozole significantly reduced the risk of distant metastases by 27% (P = 0.001) in the HR+ population, and another analysis of the early risk of relapse in 5,980 patients, with a median follow-up of 25 months, showed that letrozole reduced distant recurrences early on [3, 20]. The recently reported analysis of letrozole-only and tamoxifen-only arms in the BIG 1-98 trial showed that the time to distant metastases advantage for letrozole was consistent with these findings from the primary core analysis [5]. These data are potentially important, because the development of distant metastases directly translates into decreased survival. The ATAC trial showed a 3% relative improvement in overall survival (OS) (P = 0.7) with anastrozole at 68 months of follow-up, while a 9% relative improvement in OS (P = 0.35) was seen with letrozole at 51 months of follow-up [3, 5].
Subset analyses of randomized trials comparing letrozole or anastrozole with either tamoxifen or placebo demonstrated differences between these AIs and suggested that specific patient populations may derive differing degrees of benefit from a particular AI. In the trial, retrospective subgroup analyses with a median follow-up of 33 months revealed no significant benefit of anastrozole over tamoxifen in patients with node-positive tumors and with prior chemotherapy [21], and these findings were confirmed in the 4-year update of the ATAC trial. Thus, the hazard ratio for risk of recurrence in patients with four or more positive nodes was 0.96 (95% CI 0.72, 1.25), and in patients with prior chemotherapy, it was 0.98 (95% CI 0.76, 1.28), indicating no differences between treatments [22, 23]. No analyses in similar subgroups were presented in the 68-month update [4].
Prospectively planned subgroup analysis revealed a benefit of letrozole over tamoxifen in patients who had received chemotherapy and in those with node-positive tumors [3, 5]. In the former subset, letrozole reduced the risk of recurrence after 5 years (hazard ratio 0.70; 95% CI 0.54, 0.92; P = 0.01). In the node-positive subset, letrozole reduced the risk of an event ending a period of DFS by 29% (hazard ratio 0.71, 95% CI 0.59, 0.85; P < 0.001). The advantage for letrozole in these patient subsets was confirmed in the recent analysis of the letrozole-only and tamoxifen-only arms of BIG 1-98 [5]. Interestingly, with longer follow-up of 51 months in this monotherapy analysis, there was an emerging benefit in the node-negative group, as letrozole reduced the relative risk of recurrence by 12% in this patient population. The MA.17 trial, evaluating the efficacy of extended adjuvant letrozole therapy, although not positive for OS in the overall population, demonstrated that OS was statistically significantly improved with letrozole among lymph node-positive breast cancer patients compared with placebo (hazard ratio 0.61; 95% CI 0.38, 0.98; P = 0.04) [24].
The question of whether one third-generation AI is superior for the adjuvant treatment of postmenopausal women with HR+ breast cancer remains, as does the question of whether there are any specific patient populations who derive particular benefit from a specific AI. Patients with early breast cancer can be assigned to risk groups on the basis of clinical and pathological characteristics. In the St. Gallen Guidelines [25], node-positive patients are considered to be in the intermediate- or high-risk group depending on the number of positive nodes and human epidermal growth factor receptor-2 (HER2) expression (see Table 1). The guidelines state that endocrine therapy with an AI is a recommended option for patients with node-positive tumors who are in the intermediate- or high-risk groups. As high-risk patients are at greater risk of relapse, a drug specifically effective in this patient population would provide the oncology community with valuable information that may alter the outcomes of these patients.
Table 1Risk categories for early breast cancer according to the St. Gallen Guidelines. Reprinted from [25], with permission from the European Society for Medical OncologyLow risk Steroid hormone receptors expression, node-negative, and all of the following features:pT ≤ 2 cmGrade 1No peritumoral vascular invasionHER2/neu gene neither overexpressed nor amplifiedAge ≥35 yearsIntermediate riskNode-negative and at least one of the following features:pT > 2 cmGrade 2–3Peritumoral vascular invasionHER2/neu gene either overexpressed or amplifiedAge < 35 yearsNode positive (1–3 involved nodes) and HER2/neu gene neither overexpressed nor amplifiedHigh riskNode positive (1–3 involved nodes) and HER2/neu gene either overexpressed or amplifiedNode-positive (4 or more involved nodes)HER2 human epithelial growth factor receptor 2pT pathological tumor size (i.e. size of the invasive component)
Is one AI superior in early breast cancer?
Microarray analysis was used to study the effects of various hormone therapies on ER+ MCF-7 cells, stably transfected with the aromatase gene (MCF-7aro cells) [26]. The study found that hormonal stimulation of gene expression can be counteracted by treatment with AIs (letrozole and anastrozole) and an anti-estrogen (tamoxifen), but that each agent had its own unique effects on gene expression (see Fig. 1), suggesting possible differences between letrozole and anastrozole [26]. Although differences between letrozole and anastrozole have been demonstrated in preclinical models, it is widely recognized that preclinical findings do not always translate into clinical results, and that comparisons in one treatment setting or subpopulation cannot be extrapolated to another. A prospective trial is therefore needed to address the question of whether one AI is superior to another.
Fig. 1Changes in inhibitor-responsive genes after treatment with letrozole, anastrozole, or tamoxifen. The Venn diagrams show the numbers of genes responsive to individual inhibitors in hormone-regulated genes. Reprinted from [26], with permission from the American Association for Cancer Research
FACE was designed to test whether there is a preferable AI for the adjuvant treatment of postmenopausal women with HR+ and lymph node-positive cancer [27]. Node-positive patients were selected, because this population has a higher risk of relapse, and recurrence events occur earlier than in node-negative patients [20, 28, 29]. Thus, conducting the FACE trial in patients with lymph node-positive early breast cancer will provide an answer more quickly than conducting a trial in a broader population that includes patients with node-negative tumors.
FACE trial design
FACE is a phase IIIb open-label, randomized, multicenter trial [30]. The primary objective of the trial is to compare DFS at 5 years for letrozole and anastrozole. Secondary objectives are to assess safety, OS, time to distant metastases, and time to contralateral breast cancer [27].
Patients
The trial is recruiting 4,000 patients from up to 250 international sites. Eligible patients are postmenopausal women with HR+ and lymph node-positive tumors who have recently undergone surgery for primary breast cancer (pathologic or clinical stage IIA, IIB, or IIIA). All patients must provide written informed consent.
HR+ tumors are defined as tumors with any detectable ER or PgR expression by institutional standards. Patients who are PgR+ and ER− are eligible for the trial. Pathologic assessment of axillary lymph nodes is determined by sentinel node biopsy and/or axillary lymph node dissection. Patients are stratified according to the number of involved lymph nodes and HER2 tumor status. Adjuvant trastuzumab is permitted in patients with HER2+ tumors. Other inclusion criteria include World Health Organization performance status of 0 or 1, lipid panel (fasting total cholesterol and triglycerides) ≤ grade 1 (National Cancer Institute Common Terminology Criteria for Adverse Events v3.0), and adequate hematologic, hepatic, and renal function.
Patients with T4 tumors, metastatic disease, contralateral breast cancer including ductal carcinoma in situ, or evidence of disease progression are excluded. Other exclusion criteria include prior neoadjuvant endocrine therapy; hormone replacement therapy (except intravaginal estradiol preparations) not stopped at least 4 weeks before randomization; adjuvant anti-estrogen therapy for > 1 month immediately following surgery, radiotherapy, and/or chemotherapy; breast cancer chemoprevention with anti-estrogens if < 18 months between stopping and diagnosis of breast cancer; and therapy with any hormonal agent, such as raloxifene, for management of osteoporosis.
Randomized trial design and treatments
Eligible patients are randomized to receive either letrozole 2.5 mg or anastrozole 1 mg daily for up to 5 years (see Fig. 2). The date of randomization must be no more than 12 weeks from completion of surgery or 4 weeks after completion of adjuvant chemotherapy. Treatment assignments are balanced based on the number of lymph nodes (1–3, 4–9, 10+) and HER2 status (positive, negative, or unknown). Treatment will commence within 30 days of randomization and following the completion of standard chemotherapy (if given) and concurrently with radiotherapy (if given). Patients receive treatment with the allocated AI for up to 5 years or until disease recurrence/relapse. Recurrence and survival will be assessed every 12 months.
Fig. 2FACE randomized trial design
Efficacy end points
The primary end point is DFS, defined as the time from the date of randomization to the date of the first documentation of re-occurrence of invasive breast cancer in local, regional, or distant sites; new invasive breast cancer in the contralateral breast; or death from any cause.
Secondary efficacy end points include OS, defined as the time from the date of randomization to date of death from any cause; breast cancer-free survival, defined as the time from date of randomization to the date of death due to breast cancer; time to development of distant metastases, defined as the time from date of randomization to the date of the first development of any recurrent or metastatic disease in sites other than the local mastectomy scar, the ipsilateral breast in case of breast conservation, or the contralateral breast; and time to development of contralateral breast cancer, defined as the time from date of randomization to the date of the first development of any disease in the contralateral breast.
Although the FACE trial, co-chaired by Drs. Ian Smith and Joyce O’Shaughnessy, is an open-label trial, analysis of the data in a blinded fashion will make the data from this trial comparable with that obtained in a single-blinded trial. Both patients and their physicians will know which drug is being taken, but the analysis of the data will be conducted blinded to study treatment. The sponsor of the trial will not have access to the database, and all efficacy analyses will be conducted by an independent academic organization (the Instituto Nazionale Tumori, Milan, Italy), which will receive the data in a blinded manner. The data will be reviewed by an Independent Data Monitoring Committee, chaired by Professor Martine Piccart. The Independent Data Monitoring Committee will then make recommendations to a Trial Steering Committee chaired by Dr. Kathy Pritchard. The Independent Data Monitoring Committee will decide when the data will be released. The final analysis will be performed after the expected total number of DFS events have occurred. This is anticipated to be 7 years after the start of the study, following an accrual period of about 2 years and a minimum of 5 years of further follow-up. There are two planned interim analyses, scheduled to occur after one third and subsequently after two thirds of the maximum number of events have been observed. The interim analyses will be conducted after 320 and 639 events, respectively, have been recorded. In addition, analyses of secondary end points will be conducted at the interim time points.
FACE is powered to detect a 3.5% absolute difference between the two treatment arms in DFS at 5 years. The 3.5% difference corresponds to a hazard ratio of 0.83 in favor of letrozole, corresponding to 5-year DFS values of 80.0% and 76.5% for letrozole and anastrozole, respectively.
Safety end points
General patient safety and drug tolerability will be evaluated. Adverse events are recorded at every visit and graded for severity using the National Cancer Institute Common Terminology Criteria for Adverse Events v3.0. A checklist of adverse events is used to solicit adverse event information from patients.
Safety analyses specifically include cardiovascular events and bone fracture events. All patients are evaluated clinically for osteoporosis and fracture risks. Bone mineral density testing is recommended at least every 2 years for all patients during study therapy by dual X-ray absorptiometry, peripheral dual X-ray absorptiometry, or ultrasound densitometry. Osteoporosis may be managed as clinically indicated using calcium supplements, vitamin D, or bisphosphonates. Measurements of fasting serum lipids are obtained at 6 and 12 months and then annually thereafter for the duration of the study treatment. Other laboratory assessments include hematology and blood chemistry.
Other head-to-head studies
Other trials that are directly comparing AIs are also under way. A randomized phase III trial [31] is comparing neoadjuvant therapy with exemestane, letrozole, or anastrozole in postmenopausal women undergoing surgery for stage II or stage III breast cancer. Another ongoing randomized trial, MA.27 [32], has been designed to compare the event-free survival of postmenopausal women with HR+ primary breast cancer when treated with exemestane or anastrozole. Results from these trials and ongoing pharmacogenomic studies [26] will also help individualize AI therapy for early breast cancer.
The study of inherited genetic polymorphisms that affect drug response and toxicity promises to help physicians individualize hormone treatment. For example, “slow metabolizers” of tamoxifen may have a worse outcome in the adjuvant setting with tamoxifen treatment than “fast metabolizers,” suggesting that these patients might be better treated with an AI [33–35]. Polymorphisms in tamoxifen metabolizing cytochrome P (CYP) 2D6 gene affect the plasma concentration of tamoxifen active metabolites: women with the CYP2D6 *4/*4 or wt/*4 genotype could have lower benefit of tamoxifen treatment and tend to have a higher risk of disease relapse [35, 36]. Genetic polymorphisms in the aromatase gene, CYP19, have recently been characterized [37]. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies revealed that polymorphisms may lead to changes in aromatase activity and altered affinity for AIs. These findings indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogen-dependent disease. Clinical trials have been initiated to study the impact of genetic differences on response to AI therapy and may eventually lead to patient-specific selection of therapy based on optimizing efficacy and toxicity.
Conclusions
Letrozole and anastrozole have both demonstrated superior efficacy compared with tamoxifen as initial therapy for early breast cancer [3, 4]. Preclinical and clinical evidence suggests that AIs do not have identical pharmacodynamic profiles, but it is not known whether one agent may be more effective as adjuvant therapy for early breast cancer. Differences in potency in preclinical studies, and the reduction in distant metastases in the BIG 1-98 study, suggest the potential for clinical efficacy differences between AIs. Based on the results of these trials, international guidelines now recommend adjuvant hormone therapy with an AI [9, 25] in patients with an increased risk of early recurrence. The FACE trial is addressing an important medical question in the oncology community: whether or not letrozole offers greater clinical benefit to postmenopausal women with HR+ early breast cancer at increased risk of early recurrence compared with anastrozole. Results from the FACE trial may refine the treatment strategies for treating breast cancer in postmenopausal women.

letrozole
aromatase inhibitors
anastrozole
tamoxifen
breast cancer
femara


Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm.
Introduction
When light energy is absorbed in vivo by purple bacterial light-harvesting (LH) complexes it is rapidly transferred to the reaction centers (RC) where the light energy is efficiently used to drive chemical reactions (Ke 2001). In most types of purple bacteria there are two types of antenna complexes: peripheral LH2 complexes and the LH1 complexes (Ke 2001). The structure of the LH2 complex of Rhodopseudomonas acidophila strain 10050 has been resolved to a resolution of 2.0 Å (McDermott et al. 1995). This LH2 complex consists of a ring of nine heterodimeric subunits. However, such high-resolution structure has not been determined for the LH1 complex yet. There are, however, low-resolution projection structures produced by transmission electron microscopy (TEM) (Karrasch et al. 1995) of two-dimensional (2D) crystals of the LH1 complex and a 4.8 Å X-ray crystal structure of the LH1-RC core complex (Roszak et al. 2003). TEM analysis of the LH1 complexes revealed two types of complex, monomeric complexes from Rhodospirillum rubrum (R. rubrum) (Karrasch et al. 1995) and dimeric complexes from Rhodobacter sphaeroides (R. sphaeroides) (Jungas et al. 1999). The recent crystal structure of the LH1-RC ‘core’ complex from Rhodopseudomonas palustris (Rps. palustris) reveals that the LH1 complex surrounds the contours of the RC so that the ‘core’ complex has an overall oval rather than a circular shape (Roszak et al. 2003). This structure showed the RC surrounded by the LH1 complex which consisted of 15 pairs of transmembrane helical α- and β-polypeptides and their coordinated BChls. The complete closure of the RC by the LH1 is prevented by a single transmembrane helix called W. Atomic force microscopy has also been used to observe antenna complexes in both natural and reconstituted membranes (Scheuring et al. 2001, 2003, 2004; Fotiadis et al. 2004; Bahatyrova et al. 2004a, b; Stamouli et al. 2003). Scheuring et al. observed the LH1 complex as a minor component together with the major LH2 complexes from Rubrivivax geratinosus (Scheuring et al. 2001) in reconstituted membranes and intact LH1-RC core complexes in native photosynthetic membranes (chromatophore) from Rhodopseudomonas viridis (Scheuring et al. 2003) and Rhodospirillum photometricum (Scheuring et al. 2004). Fotiadis et al. observed the LH1-RC core complex from R. rubrum in membranes formed from Escherichia coli lipids (Fotiadis et al. 2004). Bahatyrova et al. showed that the LH1 complexes of a mutant lacking the RC from R. sphaeroides which form circular, elliptical, and even polygonal ring shapes as well as arcs and open rings (Bahatyrova et al. 2004b) and that the LH1 complexes are positioned to function as an energy collection hub from the LH2 complexes in native membranes (Bahatyrova et al. 2004a).
Our understanding of charge separation and energy transfer in these LH2 and LH1-RC core complexes has enabled the first steps to be taken towards generating artificial systems that convert light energy into usable electrical current. Previous attempts to produce an artificial, energy-converting electrode system used either the LH1 complexes (Ogawa et al. 2002) or RC (Blankenship et al. 1995) immobilized on the electrodes. Until now, there have only been a few attempts to immobilize intact ‘core’ complexes, consisting of both the LH1 and the RC components together, onto an electrode (Ogawa et al. 2004; Das et al. 2004).
We have recently developed a procedure to create a self-assembled monolayer (SAM) of reconstituted LH1 complexes on a transparent indium tin oxide (ITO) electrode modified with 3-aminopropyltriethoxysilane (APS) using electrostatic interactions or hydrogen bonding between the electrode surface and the anionic LH1 polypeptides at pH 8.0 (Ogawa et al. 2002). The near infra-red (NIR) absorption spectrum showed that the LH1 complex was stable when immobilized onto the electrode. Our current work extends this approach to the native LH1-RC core complexes. LH1-RC ‘core’ complexes isolated from R. rubrum and Rps. palustris were successfully assembled on an ITO electrode modified with APS (APS-ITO). Efficient energy transfer and photocurrent responses could be observed upon illumination at 880 nm.
Materials and methods
Growth of R. rubrum and Rps. palustris bacterium
The photosynthetic bacteria, R. rubrum strain S1 and Rps. palustris strain 2.1.6 were grown anaerobically in the light in modified Hutner’s media as previously described (Roszak et al. 2003; Visschers et al. 1991).
Purification of the LH1 complex of R. rubrum
Chromatophores of R. rubrum were prepared as previously described (Visschers et al. 1991). Carotenoid was extracted from chromatophores using benzene. Usually approximately 30 mM OG (1 mM Tris, pH 7.5) was added to dissolve the chromatophores until the near IR absorption band shifted from 873 to 820 nm. Then the resultant aqueous solution was applied to a Sephadex G-100 column (1.5 cm i.d. × 75 cm) to separate the RC and, LH1 complexes with carotenoids and subunit LH/BChl a complexes without carotenoid (B820 complexes). The RC was eluted immediately after the void volume and then the LH1 complexes with carotenoid. Finally the B820 complexes were collected. Absorbance (λmax) of the LH1 complex in the absence of carotenoid (15 mM OG at 25°C): 372 nm (0.86), 586 (0.23), 870 (1.14). Absorbance (λmax) of the LH complex in the presence of carotenoid (20 mM OG at 25 °C): 372 nm (0.86), 475 (0.19), 512 (0.22), 546 (0.19), 586 (0.235), 875 (0.9).
Isolation and purification of the core complex of R. rubrum and Rps. palustris
The LH1-RC core complexes isolated from R. rubrum and Rps. palustris were purified essentially as described previously (Roszak et al. 2003). These LH1-RC core complexes from R. rubrum and Rps. palustris were initially solubilized by the addition of LDAO to 0.4% or 1% v/v in 20 mM Tris–HCl pH 8.0, respectively. The LH1-RC core complex of Rps. palustris was then separated from the LH2 complexes by sucrose density centrifugation, and was further purified by ion exchange chromatography by DE52 cellulose column. The OD880 of the isolated LH1-RC core complexes was adjusted to 0.3.
Preparation of the core complex assembled on APS-ITO electrodes
The basic methods for this have been reported previously (Ogawa et al. 2002). Transparent indium tin oxide electrodes were cleaned by immersion in piranha solution (H2O2:H2SO4 = 3:7). We obtained APS-ITO electrodes by reacting 3-aminopropyltriethoxysilane with the surface of ITO electrodes in dry benzene at 80–90°C for 4 h. The OD880 of the isolated core was adjusted to 0.3. APS-ITO electrodes were immersed in the LH complex and the core complex solutions in Tris–HCl pH 8.0 for 6 h at 4°C, and then rinsed with Tris–HCl pH 8.0. The core complexes were immobilized, as a self-assembled monolayer on an APS-ITO electrode.
Near IR, FT-IR, and fluorescence spectra
Near IR spectra were recorded with Hitachi U-2000 and U-3500. Fluorescence spectra were measured with a Nippon Roper fluorometer by using a halogen tungsten light bulb (TS-428 DC), a single monochromator (SP-150M) for selection of the excitation wavelength, a monochromator (SP-306) and a CCD detector (Spec 10-100 BR/LN) to detect the emitted fluorescence. The slits were set at 0.50 mm for the LH1 complexes in OG. Slits were set at 1.00 mm for the LH1 complex on the electrode. The samples were measured at 25°C. FT-IR spectra were recorded with Perkin-Elmer Spectrum 2000.
Photocurrent measurements
Photocurrents were measured at −0.2 V (versus Ag/AgCl) in a home made cell that contained three electrodes; an APS-ITO electrode incorporating the core complex as a working electrode, an Ag/AgCl (saturated KCl) as a reference electrode, and a platinum flake as a counter electrode. The working electrode was illuminated with a halogen lamp unit, AT-100HG, through a monochromator, SPG-120S (Shimadzu). The solution consisted of 0.1 M phosphate buffer (pH 7.0), containing 0.1 M NaClO4 and 5 mM methyl viologen.
Results and discussion
Figure 1 shows the NIR absorption spectra of the isolated R. rubrum (a) and Rps. palustris (b) core complexes in 20 mM Tris–HCl buffer pH 8.0 OG micelle (dotted line) and assembled onto an APS-ITO electrode (solid line), respectively. These spectra show that these core complexes have the absorption maximum at 880 nm with two smaller peaks at 800 and 760 nm. The former peak is attributable to the overlap of bacteriochlorophyll a (BChla) in the LH1 complex (880 nm) and the reaction center BChla dimer ‘special pair’ (870 nm) and the latter two peaks to the BChla called ‘accessory’ (800 nm) and bacteriopheophytin (760 nm) in the RC, respectively (Blankenship et al. 1995). The NIR absorption spectra of these core complex on the electrode indicate that these complexes were not denatured when assembled onto an APS-ITO. In the previous study it was apparent that when the RC of R. rubrum was assembled, by itself, on the electrode it was relatively labile (Matsumoto et al. 1999). In present study, the complete core complex proved to be quite stable, when assembled onto the electrode. The enhanced stability of the RC surrounded by the LH1 complex probably results from supportive interactions between the two complexes.
Fig. 1NIR absorption spectra of the isolated R. rubrum (a) and Rps. palustris (b) core complexes in 20 mM Tris–HCl buffer pH 8.0 OG micelle (dotted line) and assembled onto an APS-ITO electrode (solid line)
Table 1 shows the NIR absorption and fluorescence bands of the LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustris in OG micelles and on an APS-ITO electrode. The fluorescence bands of these core complexes on the electrode are identical to those in the OG micelles, again indicating that these complexes are stable when assembled onto the APS-ITO electrode. Interestingly, when illuminating at 880 nm the fluorescence emission of BChla molecules in the LH complex of R. rubrum on the APS-ITO was strongly quenched, due to the presence of the RC of R. rubrum. This indicates that an efficient energy transfer from BChla in the LH1 complex to the RC in the core complex is still occurring on the electrode (data not shown) (Ogawa et al. 2004). FT-IR spectra of the LH complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustris assembled on the APS-ITO show the absorptions at 1650 and 1550 cm−1. These bands can be assigned to the amide I (C=O stretching vibration) and amide II (N–H deformation vibration) bands of α helical conformation, respectively (Miura et al. 1998). These results indicate that the LH polypeptides are in the same α helical configurations on the ITO electrode as in OG micelles (Parkes-Loach et al. 1988; Kashiwada et al. 2000).
Table 1NIR absorption and fluorescence bands of the LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustrisIn OG micelleOn APS-ITOAbsorbance (nm)Fluorescence (nm)Absorbance (nm)Fluorescence (nm)R. rubumLH1870890870890R. rubumLH1-RC880900880900Rps. palustrisLH1-RC878900878900
In summary, all of these results indicate that the LH1 and LH1-RC core complexes were not denatured by binding to the APS-ITO surface. Similar results were obtained using Langmuir–Blodgett (LB) films to lay down LH1 complex membranes as a layer on a glass substrate (Iida et al. 2000). The LH1 complex of R. rubrum and the LH1-RC core complexes of R. rubrum and Rps. palustris on the APS-ITO electrode were stable enough to handle at 4°C in the dark condition least 24 h.
Figure 2 shows the time course of the photocurrent generated from the LH1-RC core complex, LH1 complex or the RC of R. rubrum assembled onto an APS-ITO when the electrode was illuminated with a pulse of light at 880 nm. It is clear in Fig. 2 that an enhanced photocurrent was observed for the LH1-RC core complex. In contrast no photocurrent was observed for either LH1 complex or the RC. Under our experimental conditions a cathodic photocurrent was observed, implying that one-way electron transfer from pigments in the LH1-RC core complex to methyl viologen was occurring as shown in Fig. 3 (Nagata et al. 2003; Imahori et al. 2000).
Fig. 2Time course of the photocurrent of the LH1-RC core complex, LH1 complex or the RC complex of R. rubrum on an APS-ITO electrode when the electrode is illuminated with pulsed light (880 nm) firing continuously for 30 sFig. 3Schematic drawing of LH1-RC core complexes on an APS-ITO electrode generated cathodic photocurrent which shows the electron flow from the complex to methyl viologen (left) according to the cathodic photocurrent as shown in Fig. 2. Energy diagram for cathodic photocurrent generation by the LH1-RC core complex (right)
Figure 4 shows excitation spectrum of the photocurrent density (dots) and the NIR absorption spectra (solid line) from R. rubrum (a) and Rps. palustris (b) LH1-RC core complexes assembled onto an APS-ITO, respectively. These photocurrent responses showed a maximum at the wavelength corresponding to the absorption bands of the complex. Interestingly, an enhanced photocurrent was observed especially upon illumination at 880 nm for both R. rubrum (a) and Rps. palustris (b) LH1-RC core complexes. The quantum yield of the photocurrent was 0.05% for both the R. rubrum and Rps. palustris complexes (Imahori et al. 2000). When the LH1 complex of R. rubrum alone, was immobilized on the electrode, the observed photocurrent was mainly generated by light absorbed at 770 nm, i.e. from monomeric BChla (Nagata et al. 2003). Furthermore, when the RC complex of R. rubrum only was immobilized on the electrode, an efficient photocurrent was not observed upon illumination at 880 nm as shown in Fig. 2. Thus, the enhanced photocurrent observed at 880 nm in the assembled LH1-RC core complex can be ascribed to energy transfer from the LH1 to the RC and then electron transfer from the electrode to the RC as shown in Fig. 3 (Ogawa et al. 2004). This data indicates that the LH1-RC core complex was well organized on the ITO and the photocurrents were driven by light that was initially absorbed by the LH components.
Fig. 4Photocurrent density (dots) and NIR absorption spectrum (solid line) of LH1-RC core complexes form: (a) R. rubrum and (b) Rps. palustris assembled on an APS-ITO electrode
In conclusion, the SAM method is clearly successful in allowing assembly of functional LH1-RC core complexes on the electrode. This has been confirmed by NIR absorption spectroscopy, demonstrating that the photocurrent response, which is derived from electron transfer between the RC and the electrode, is enhanced by illumination at 880 nm.

light harvesting-reaction center complex


Perceived work-related stress and early atherosclerotic changes in healthy employees

Objective This study was conducted to investigate the relationship between perceived work-related stress and preclinical atherosclerosis.
Introduction
Stress can no longer be recognized as a problem of an individual only. Due to its versatile presence in every human’s life, often resulting in health complications, stress has become a social problem and the role of work-related stress in this puzzle should not be underestimated. Coronary heart disease (CHD) is on the list of stress-related health problems.
Atherosclerosis, the basis of CHD, develops in a long-lasting, multifactorial and complex process. In the etiopathogenesis of atherosclerosis, apart from traditional factors like lipids, carbohydrate metabolic disorders, hypertension and lifestyle (smoking, alcohol use and limited physical exercise), immunological disorders, as a consequence of reciprocal interactions in the psycho-neuro-immunological system, have been recently brought up (George and Shoenfeld 1997; Bednarska-Makaruk and Pasierski 2000; Kiecolt-Glaser and Glaser 1991; Amengual et al. 2001; Patryka et al. 2001; Jędryka-Góral et al. 2002; Jędryka-Góral 2003; Dinan 2005). Early atherosclerosis can even occur in young healthy people (Pasierski 1999).
For a long time now, occupational medicine has been interested in the relationship between work-related stress and CHD. Studies undertaken in this field have been based on western as well as eastern european populations (Jonsson et al. 1995; Hammer et al. 1998; Bosma et al. 1998, Kristenson et al. 1998; Kivimaki et al. 2002; Smith et al. 2005; Malinauskiene et al. 2005). Although there is a large body of literature on clinically overt CHD and its relation to work-related stress, there have been very few studies on preclinical CHD in those circumstances (Lynch et al. 1997; Nordstrom et al. 2001; Rosvall et al. 2002; Hintsanen et al. 2005).
The current study aimed to fill this gap and to assess the relationship between work-related stress and preclinical atherosclerotic changes in healthy employees. We considered carotid artery intima-media thickening and/or plaque presence as a surrogate of coronary atherosclerosis. To detect these changes “gold-standard” methodology, i.e., B-mode carotid ultrasound examination, was applied. Because of the complex nature of the pathogenesis of CHD, other contributing factors have not been disregarded.
Methods
Organization of the study
First, all participants took part in a psychological study in which the level of stress and coping were evaluated with relevant questionnaires (see below). Then, the participants consecutively presented themselves for a clinical visit in which a medical examination took place and a blood sample was taken. Tests for biochemical parameters (see below) were performed on the same day as the blood sample was taken, according to routine laboratory procedures. Sera for immunological parameters (see below) were collected and stored according to international laboratory standards, then tested in runs (up to 40 sera per day) to minimize day-to-day variations.
Participants
One hundred healthy managers and 50 office workers (F:M = 50:50) aged 35–65 were studied. All of them were investigated for the presence of individual (age, obesity, hypertension, diabetes, smoking, alcohol use and low physical activity) and family (obesity, hypertension, diabetes, heart attack and stroke) risk factors for atherosclerosis. The studied individuals’ detailed characteristics are presented in Table 1.
Table 1Characteristics of studied groupsManagers (n = 100)Office workers (n = 50)Total (n = 150)Number50 (50%)25 (50%)75 (50%) Females Males50 (50%)25 (50%)75 (50%)Age47.2 ± 6.850.16 ± 6.4648.68 ± 6.63 Mean (years)  Minimum343534.5 Maximum656464.5Individual risk factors26 (26%)23 (46%)49 (32%)  Hypertension  Obesity19 (19%)17 (34%)36 (24%) Diabetes1 (1%)1 (2%)2 (1.3%)Alcohol use Every day3 (3%)03 (2%)  2–3×a week23 (23%)2 (4%)25 (16.7%)  Less69 (69%)47 (94%)116 (77.3%)  Never5 (5%)1 (2%)6 (4%) Smoking cigarettes49 (49%)30 (60%)79 (52.7%) Hormone replacement therapy8 (8%)4 (8%)12 (8%)Physical activity  2–3×a week26 (26%)12 (24%)38 (25%)  1×in 7–10 days32 (32%)14 (28%)46 (30.7%)  Less42 (42%)24 (48%)66 (44%)Family risk factors41 (41%)31 (62%)72 (48%) Hypertension Obesity45 (45%)23 (46%)68 (45.3%) Diabetes31 (31%)12 (24%)43 (28.7%) Heart attack36 (36%)21 (42%)57 (38%) Stroke30 (30%)17 (34%)47 (31.3%)
Instruments
Work-related stress was operationalized as perceived pressure caused by inappropriate working conditions. It was measured by the “Sources of pressure in your job” scale that is a part of the short version of the Occupational Stress Indicator (OSI-2) by Cooper et al. Widerszal-Bazyl’s (2001) Polish adaptation of OSI-2 was used in the study. The scale consists of 40 items on potential sources of pressure at work; respondents are required to rate each source on six-point response scales, in which 1 = it is very definitely not a source (of pressure) to 6 = it is very definitely a source. Those 40 items were divided into eight subscales concerning the following stress factors: workload (e.g. “Having to work very long hours”, “Taking work home”), relationships (e.g. “Inadequate guidance and backup from superiors”, “Feeling isolated”), home–work balance (e.g. “Pursuing a career at the expense of home life”), recognition (e.g. “Underpromotion—working at a level below my level of ability”), organizational climate (e.g. “Characteristics of the organization’s structure and design”, managerial role (e.g. “Managing or supervising the work of other people”), personal responsibility (e.g. “Having to take risks”), hassle at work (e.g. “Attending meetings”). The scale made it possible to assess the global level of work-related stress (the sum of scores for all items) as well as to assess the level of a given kind of work stress, e.g. workload (the sum of scores for items related to workload). The reliability of the Polish version of the total scale was very high: Cronbach’s α = 0.95. It was also satisfactory for most subscales: workload α = 0.95, relationships α = 0.89, home–work balance α = 0.81, recognition α = 0.75, organizational climate α = 0.63, managerial role α = 0.57, personal responsibility α = 0.78, hassle at work α = 0.54. (Widerszal-Bazyl 2001).
Coping was measured with the “How you cope with the stress you experience” scale that is also part of OSI-2, too. It consists of 10 items on potential coping strategies; respondents are required to rate the extent to which they actually use them as ways of coping with stress. Answers are given on six-point scales, from 1 = I never use it to 6 = I use it very extensively. Some coping strategies included into the scale dealt with coping through control (e.g. “Effective time management”), and some with coping through social support (e.g. “Talk to understanding friends”). The reliability of the Polish version of the total coping scale was Cronbach’s α = 0.70, and reliability of its subscales: 0.70 (for coping through control) and 0.50 (for coping through social support). Therefore, the total index of coping was used in the analysis, as well as the index of coping through control.
Prior to the study, appropriate approval from the Local Ethics Committee was obtained. Participants were provided with detailed written information on the study’s objectives and the methods that were going to be used; subsequently they signed their informed consent.
Serum levels of biochemical (total cholesterol, LDL, HDL, TG, glucose) and serological risk factors of atherosclerosis (anticardiolipin, anti-β2 GPI, anti-oxLDL, anti-HSP and anti-hsCRP antibodies) were evaluated.
Anticardiolipin antibodies Patients’ sera were tested with ELISA for the presence of anticardiolipin antibodies, according to the modified Harris method (Luft et al. 1990). The results were presented as OD units. Reference ranges of aCL were established by studying sera from 100 healthy donors. The cut-off value was established as OD = x + 4SD estimated for sera of healthy blood donors; it was 0.109 for the IgG class and 0.156 for the IgM class.
Anti-β2 glycoprotein I (β2 GPI) antibody levels were measured with a commercial DIASTAM—FBGP 200 from Axis-Shield (Cat. No. FBG200) test. The results were expressed as OD units. Reference ranges of anti-β2 glycoprotein I antibodies were established by studying sera from 152 healthy Caucasian donors (Axis-Shield Diagnostics Ltd. examination). The cut-off value was established as OD = 0.195.
Anti-HSP antibody levels (IgG class) were estimated with non-commercial methodology, after necessary modification, according to Tsoulfa et al. (1989), with the use of McLean reagents. The cut-off value was established as x + 4SD estimated for sera of healthy blood donors; it was OD = 0.423. The estimation of IgM and IgA class antibodies was performed in the same manner.
Anti-oxLDL IgG antibody levels were measured with ELISA with the use of a commercial test from OLAB (Cat. No. BI-20032). The cut-off value was established as 200 arbitrary units.
hsCRP concentration was measured with ELISA with the use of commercial tests from EUCARDIO Laboratory Inc. (Cat. No. ZZ C4011E). The cut-off value was established as 4000 ng/ml.
Intima-media thickness (IMT) and atherosclerotic plaque in carotid arteries were searched using a computer analysis of B-mode ultrasound images. Both carotid arteries were measured with a 7.5-MHz linear-array transducer, part of Hewlett Packard Sonos. Plaque was defined as intima-medial thickening larger than 50% in comparison to the surround area (Pignoli et al. 1986)
Statistical analysis of data was performed with SPSS v. 11.5. Means comparisons were carried out with a t test for independent groups. In bivariate correlation analysis Pearson’s r coefficients were calculated. The level of significance was set at α < 0.05.
Results
Assessment of work-related stress
The level of work-related stress measured with OSI-2’s Sources of pressure scale was as follows: managers—mean = 154 (σ = 25), for office workers—mean = 145 (σ = 28), for both groups—mean = 151(σ = 25). According to Polish sten norms, the sten scale (“sten” stands for standard ten) consists of ten units. Each unit equals 0.5 of a standard deviation. It is, assumed that stens five and six means average results, stens seven to ten high results, and stens one to four low results (Canfield 1951). Developed for Polish managers (Widerszal-Bazyl 2001), the managers’ mean falls on sten six, whereas the office workers’ mean falls on sten five (Table 2). As a rule, stens five and six are interpreted as average results.
Table 2Results of psychological examinationStudied groupsWork-related stressCopingMeanStensMeanStensManagers154664056Office workers145553855Total151654056
Assessment of coping
The level of coping measured with OSI-2’s Coping scale was as follows: managers—mean = 40, office workers—mean = 38, both groups—mean = 40. According to Polish sten norms the managers’ mean falls on sten five for men and on sten six for women. The office workers’ mean falls on sten five (for both genders). Thus, it can be said that the coping results were in the average range.
Laboratory findings
The abnormality most frequently disclosed in biochemical findings were elevated levels of total cholesterol, LDL and triglycerides (in 52, 39 and 22% of the studied individuals, respectively). Decreased levels of HDL and increased levels of glucose were less frequent and were found in 12% of cases each.
Of the immunological parameters studied, oxLDL antibodies were most frequently found (in 26% of cases), followed by an elevated level of hsCRP (in 11% of cases). In a few cases antibodies to β2 GPI and HSP-65 (IgG class) were found (in 1% and 3% of studied cases, respectively). None of the studied individuals was positive for aCl. For six sera which were borderline or positive for HSP-65 IgG class, IgM and IgA class antibodies were searched. Only in three cases were the results weakly positive (two in IgM and one in IgA class).
Ultrasound examination of carotid arteries
Changes in the ultrasound examination of carotid arteries confirming the presence of early atherosclerosis are shown in Table 3. Plaque was found in 43 (29%) cases: in 26 managers (26%) and in 17 office workers (34%); groups did not differ statistically. The distribution of the mean values of the IMT measure was slightly skewed to the right. The mean values of IMT in managers and office workers were 0.0620 ± 0.014 and 0.0610 ± 0.012 mm, respectively; groups did not differ statistically.
Table 3Results of ultrasound examination of carotid arteries in studied groupsManagers (n = 100)Office workers (n = 50)Total(n = 150) Number of individuals without changes74 (74%)33 (66%)107 (71%) Number of individuals with plaque26 (26%)17 (34%)43 (29%) Intima-media measurement values 0.0620 ± 0.0140.0610 ± 0.0120.0618 ± 0.013
Correlation between IMT and the presence of plaque in carotid arteries with atherosclerosis risk factors
Positive correlation was found between IMT and age (r = 0.42; P < 0.001), diastolic blood pressure (r = 0.20; P < 0.05), LDL (r = 0.28; P = 0.001), anti-HSP antibodies (r = 0.24; P < 0.05), whereas negative correlation was revealed with aCl—IgG (r = –0.20; P < 0.05) and global work-related stress level (r = −0.26; P < 0.01). Among the stressors studied, statistical significance was shown for interpersonal relations (r = −0.23; P < 0.00), work−home balance (r = −0.19; P < 0.05), managerial role (r = −0.24; P < 0.01) and organization climate (r = −0.22; P < 0.01). The presence of plaque correlated positively with age (r = 0.42; P < 0.001), smoking (r = 0.18; P < 0.05), LDL (r = 0.30; P < 0.01) and negatively with global work-related stress level (r = −0.28; P < 0.01). Statistical significance was shown for the following stressors: work load (r = −0.24; P < 0.01), interpersonal relations (r = −0.18; P < 0.05), work−home balance (r = −0.33; P < 0.01) and responsibility (r = −0.25; P < 0.01) (see Table 4).
Table 4Correlations between IMT and the presence of plaque in carotid arteries and atherosclerosis risk factors (Pearson’s r)Risk factorIMTPlaqueAge 0.42**0.43**BMI 0.120.04Systolic blood preasure 0.130.06Diastolic blond preasure 0.20*0.10Cigarette smoking 0.130.18*Physical activity−0.020.07LDL0.28**0.30**HDL −0.16−0.05Triglycerides0.04 0.05Glucose 0.080.02hsCRP 0.050.02Anti-HSP 0.160.19aCl–IgG −0.20*−0.10aCl–IgM −0.14−0.15Anti-oxLDL −0.10−0.17Workload −0.15 −0.24**Relationships−0.23** −0.18*Home–work balance −0.19*−0.33**Managerial role −0.24*−0.14Personal responsibility −0.14−0.25**Hassle at work −0.11−0.14Recognition −0.25**−0.15Organizational climate −0.22**−0.15Total index of coping −0.12−0.01Coping through control −0.050.02Global level of stress−0.26**−0.28*** Correlation is significant at 0.01 (2-tailed)** Correlation is significant at 0.05 (2-tailed)
Correlation between LDL/smoking and global stress level
Pearson’s analysis showed a negative relation between LDL and global stress level (r = −0.17; P < 0.05), and between smoking and global stress level (r = −0.17; P < 0.05).
Work-related stress, coping, lifestyle and IMT
Pearson’s analysis was performed in two triangles: (a) work-related stress–coping–IMT, and (b) work-related stress–healthy lifestyle–IMT. No statistically significant relations were shown either between work-related stress and coping, or between coping and IMT (P > 0.05), or between work-related stress and healthy lifestyle (no smoking, no excessive use of alcohol, high physical activity), or between healthy lifestyle and IMT (P > 0.05).
Discussion
A multidisciplinary approach to assessing risk factors for atherosclerosis was considered by us in an earlier study (Jędryka–Góral et al. 2006). On the basis of a deep multilevel analysis we were able to show that in healthy individuals, as in CHD patients, individual and biochemical risk factors were likely to play a leading role in the development of early atherosclerosis. Psychosocial stress should also be considered; however, inflammatory-immunological factors have not been found to be an independent predictor.
As inflammatory-immunological factors, antibodies to oxLDL and HSP-65, hsCRP as well as anticardiolipin and anti-β2 GPI antibodies were considered. In the extensive literature of the subject, these parameters are recognized as directly involved in the pathogenesis of atherosclerosis (George and Shoenfeld 1997; Kiecolt-Glaser, Glaser 1991; Amengual et al. 2001; Patryka et al. 2001; Jędryka-Góral et al. 2002; Jędryka-Góral 2003).
The present study was undertaken to extend the investigation of psychosocial stress and atherosclerosis. We looked in depth for correlation between preclinical atherosclerosis and work-related stress; correlations with other risk factors of CHD were done in parallel.
As expected, we found correlation between IMT and age, diastolic blood pressure, LDL and anti-HSP antibodies, and correlation between plaque and age, smoking and LDL.
Surprisingly, our results revealed that early atherosclerotic changes negatively correlated with the level of global job strain and some of its components (interpersonal relations, work–home balance, managerial role, organization climate—for IMT; work load, interpersonal relations, work–home balance, responsibility—for plaque).
Ambiguous results have been previously reported in other, although very few, studies where work, psychosocial factors and carotid atherosclerosis were studied with ultrasound. Rosvall et al.’s (2002) hypothesis that work-related stress characterized by high psychological demands and low decision latitude was associated with increased carotid atherosclerosis could not be confirmed either for women or for men. On the other hand, Lynch et al.’s (1997) prospective study showed that men who experienced work-related stress (demanding work and low economic rewards) had significantly greater progression of carotid atherosclerosis than more advantaged men. Hintsanen et al. (2005) provided data that job strain (a joint effect of job demands and job control) was associated with increased IMT in men but not in women. Similarly, Nordstrom et al. (2001) showed that in men with greater work-related stress (workplace demands and intrusion of work concerns into home life) the risk of focal lesions or intima-media thickness in coronary artery increased, whereas in women stress was not related to the prevalence of early atherosclerosis. Hlatky et al. (1995) disclosed that job strain (high psychological demands and low decision latitude) did not correlate with the presence of coronary disease in angiography, either for men or for women.
By searching for a relationship between work-related stress and atherosclerosis, we wanted to prove that the correlation found between early atherosclerotic changes and LDL and smoking is, at least partly, caused by a higher stress reaction in the studied individuals. It was assumed that individuals with a level of high stress undertook unhealthy behaviour: smoking or excessive food intake. However, for both LDL and smoking the correlation with stress was negative. One should keep in mind that only 39% of the studied individuals had elevated levels of LDL and 47% reported smoking. Therefore, these results should be interpreted causally.
We explored in depth the negative correlation we found between IMT and the presence of plaque, and the level of work-related stress. Being aware that OSI-2 measured perceived stress, another hypothesis was formulated; persons with a high level of work-related stress (conscious stress) undertake preventive activities at the level of coping or healthy lifestyle.
Perceived work-related stress as a risk factor for a clinically overt CHD has been investigated in multiple studies. Some prospective studies showed an association of work-related stress with an incidence of CHD (Bosma et al. 1998; Kivimaki et al. 2002; Kivimaki et al. 2005); whereas others did not (De Bacquer et al. 2005; Rosvall et al. 2002; Eaker et al. 2004). The largest INTERHEART study covering 11 119 cases and 13 648 controls from 52 different countries all over the world confirmed this association with regard to work, home, financial and major life stress (Rosengren et al. 2004). There are studies which, indicated that perceived work-related stress had profound impact on internal organs, sensual organs, the locomotor system and skin health problems (Cheng et al. 2001; Lindgren et al. 2002).
In the literature of the subject, coping is closely linked to stress. The most common meaning of coping is, an effort to solve problems and to seek reduction of tolerance to stress. The CATS theory (cognitive activation theory of stress) offered a new approach to coping (Ursin, Eriksen 2004; Eriksen et al. 2005). This theory assumed two cognitive reformulations of the learning theory (stimulus–stimulus learning = classical conditioning and response learning = instrumental conditioning). Both reformulations are essential to understanding the relationship between learning, activation and relations between stress and health. The stress response (an alarm in the homeostatic system) results in behaviors that aim to cope with a situation. The level of alarm depends on what outcome of a stimulus is expected and the specific responses available for coping. Response outcome expectancy might be positive (coping), negative (hopelessness) or none (helplessness). Hence, coping defined as acquired positive outcome expectancy has some predictive value for stress and health.
According to Weidner and Cain (2003) research on how people cope with stress situations disclosed avoidant mechanisms (denial, distraction, excessive alcohol consumption) in men, whereas more cardioprotective strategies (depression, asking for help)—in females.
Stress management goes far beyond coping strategies. Toobert et al.’s (2002) results disclosed success of a complex intervention program to reduce CHD risk, which included improvement of diet, stress management, social support, smoking and physical activity.
In our study on preclinical atherosclerosis and work-related stress, we were not able to confirm the hypothesis about the above-mentioned prophylactic activities. An analysis of correlation showed no significant relations between work-related stress and coping, between coping and IMT, between work-related stress and healthy lifestyle, or between healthy lifestyle and IMT.
A relatively novel conception was presented by de Lange et al. (2005), who claimed that the relationship between stressful work and psychological well-being may be reciprocal. Their results showed that mental health might influence employees’ perceived work characteristics. It is likely that the employees in our study had been recruited from that part of working society who felt fit to undertake ambitious tasks of managers and office workers and who developed job adaptive mechanisms over time.
The most probable interpretation of the negative correlation between perceived work- related stress and preclinical atherosclerosis is that in the case of individuals with a low level of perceived work-related stress, somatization of stress took place, i.e., stress is not perceived at the conscious level but it leads to somatic effects (e.g., IMT).
We are aware of some limitations of our study. Firstly, the results regarding work-related stress were based on self-reported data, which per se could always be a matter of some bias. Secondly, the number of employees studied was limited to 150. Voluntary participation of employees and fully unselected method of recruitment can guarantee the objectivity of the results. Thirdly, it was a cross-sectional study. A prospective type of research on risk assessment is preferable nowadays. However, our study did not aim to assess risk of CHD but to find correlation between work-related stress and early atherosclerosis, so we believe the measure taken for this purpose was appropriate.
We are more than convinced that to achieve full understanding of negative correlation between work-related stress and early atherosclerosis requires further interdisciplinary studies and we would be happy to continue exploration into this intriguing field.

work-related stress
early atherosclerosis
carotid arteries