Effect modification by ferritin on the relationship between inflammation and arterial stiffness in hypertensive patients with different glucose tolerance


 Backgroud: Ferritin, a crucial element for iron homeostasis, is associated with chronic diseases characterized by subclinical inflammation such as essential arterial hypertension and type 2 diabetes mellitus (T2DM), showing a prognostic value in different clinical settings. We investigated whether ferritin is associated with arterial stiffness (AS), an early indicator of atherosclerosis, and if it could act as effect modifier on the relationship between inflammation and AS in hypertensive patients with different glucose tolerance.Methods: We enrolled 462 newly diagnosed untreated hypertensive (HT) patients. All subjects underwent an oral glucose tolerance test. Insulin sensitivity was assessed by MATSUDA index and ferritin levels were estimated by immunoradiometric assay. AS was definied by carotid-femoral pulse wave velocity (PWV).Results: Out of 462 patients, 271 showed normal glucose tolerance (HT/NGT), 146 impaired glucose tolerance (HT/IGT) and 45 were diabetic (HT/T2DM). Iron levels significantly decreased and transferrin and ferritin significantly increased from the first to the third group. PWV values were significantly higher in HT/IGT and HT/T2DM patients. PWV was related directly with ferritin, high sensitivity C reactive protein (hs-CRP), transferrin, and inversely with MATSUDA index. Ferritin resulted the strongest determinant of PWV explaining a 14.9% of its variation; moreover it was a strong modifier of the relationship between hs-CRP and PWV. The estimated augmentation in PWV portended by a fixed increase in hs-CRP, was higher across increasing values of ferritin.Conclusion: Ferritin represents an independent predictor of AS in our study population and a strong effect modifier on the relationship between inflammation and PWV.

Conclusion: Ferritin represents an independent predictor of AS in our study population and a strong effect modifier on the relationship between inflammation and PWV.

Background
Iron plays a pivotal role in preservation of the biological systems but it is also true that an excess of this cation may lead to reactive oxygen species (ROS) production thus promoting cellular and tissue damage [1,2].
Ferritin is an ubiquitous protein which represents not only a crucial element of iron homeostasis regulation but also the most used biomarker of iron deficiency [3]. Moreover, serum ferritin is a well-known acute phase protein reflecting the degree of acute and chronic inflammation and compelling evidence suggest a potential active role of ferritin in chronic inflammatory diseases [4]. Accordingly, several studies report a direct association between serum ferritin levels and chronic inflammation of mild degree [5][6][7][8][9]. In particular, serum ferritin levels resulted to be directly related to insulinresistance and incident risk of type 2 diabetes mellitus (T2DM), independently of traditional risk factors [5,6], especially when high-sensitivity C reactive protein (hs-CRP) is increased [7].
Furthermore, in various populations it has been demonstrated that ferritin is associated with a high risk of arterial hypertension development [8,9], this latter being the most common risk factor of cardiovascular (CV) events and disability as well [10].
It is well known that arterial stiffness is increased in patients with T2DM and arterial hypertension [11,12] and represents not only an early biomarker of atherosclerotic process but also an independent risk factor of CV outcomes in these conditions [13,14]. In particular, increased carotid-femoral pulse wave velocity (PWV), the gold standard parameter of arterial stiffness evaluation, reflects the disparity between peripheral and aortic blood pressure (BP). A recent meta-analysis shows that 1 m/s increase in PWV is associated with a 15% risk increase of CV mortality in about 16000 individuals followed up for a mean of 8 years [15]. Although there is some evidence of an independent association between hyperferritinemia and arterial stiffness in disparate populations [16][17][18][19], data in hypertensive patients are few.

Methods
The potential effect modification by ferritin levels on the relationship between mild inflammation and PWV in patients with various degree of glucose tolerance has not been yet completely investigated. In a large group of naive hypertensive patients with different glucose tolerance status, we investigated whether serum ferritin levels, within the normal range, are associated with arterial stiffness, independently of a series of potential confounders. Furthermore, we also tested the hypothesis that serum ferritin could act as an effect modifier on the relationship between inflammation and arterial stiffness in the same patient-population.

Study Population
The study group consisted of 462 Caucasian newly diagnosed hypertensive patients, 216 men and 246 women aged 49.6 ± 12.2 years, participating in the Catanzaro Metabolic Risk factors Study. All subjects underwent physical examination and review of their medical history. Causes of secondary hypertension were excluded by appropriate clinical and biochemical tests. Other exclusion criteria were history or clinical evidence of coronary and valvular heart disease, congestive heart failure, peripheral vascular disease, chronic inflammatory diseases, anemia, gastrontestinal diseases with malabsorption, history of any malignant disease, history of alcohol or drug abuse, liver or kidney failure and diabetes already diagnosed. No patient had ever been treated with antihypertensive drugs. All subjects underwent anthropometrical evaluation with measurements of weight, height, and body mass index (BMI). After 12-h fasting, a 75 g oral glucose tolerance test (OGTT) was performed with 0, 30, 60, 90 and 120 minutes sampling for plasma glucose and insulin. Glucose tolerance status was defined on the basis of OGTT using the World Health Organization (WHO) criteria. The Ethical Committee approved the protocol and informed written consent was obtained from all participants. All the investigations were performed in accordance with the principles of the Declaration of Helsinki.

Blood Pressure Measurements
Measurements of clinic BP were obtained in the left arm of the patients, in supine position, after 5 min of quiet rest, with a aneroid sphygmomanometer. A mean value of at least three BP readings were taken in three different visits at least 2 weeks apart. Systolic and diastolic BP were recorded at the first appearance (phase I) and the disappearance (phase V) of Korotkoff sounds, respectively. A value of clinic systolic BP (SBP) ≥ 140 mmHg and/or diastolic BP (DBP) ≥ 90 mmHg identified patients as hypertensive according to current guidelines [20]. In addition, pulse pressure (PP) was defined as the difference between SBP and DBP.

Laboratory Determinations
All blood samples were obtained after overnight fasting. Serum ferritin levels (10-290 ng/ml) were measured using an immunoturbidimetric assay (Roche Diagnostics, Indianapolis, IN, USA). The serum iron (50-150 µg/dl) was measured with a colorimetric method and transferrin (2-4 g/l) by a nephelometric assay, while the percentage of saturated transferrin iron binding capacity (TIBC) was measured using a colorimetric test (Roche Diagnostics, Cobas 8000, Switzerland). Hemoglobin was determined using an automated particle counter (Siemens Healthcare Diagnostics ADVIA® 120/2120 Haematology System, Milan, Italy).
Insulin sensitivity was evaluated using the Matsuda index [insulin sensitivity index (ISI)], calculated as follows: 10,000/square root of [fasting glucose (millimoles per liter) x fasting insulin (milliunits per liter)]*[mean glucose * mean insulin during OGTT]. The Matsuda index is strongly related to euglycemic hyperinsulinemic clamp that represents the gold standard test for measuring insulin sensitivity [21].

Arterial stiffness evaluation
All measurements were obtained using a validated system (Sphygmocor™; AtCor Medical, Sydney, Australia) that utilizes high-fidelity applanation tonometry (Millar) and an appropriate computer software for the evaluation of pressure wave (Sphygmocor™). At first, the pressure calibration was achieved by the non-invasive automatic recording of supine brachial artery BP in the dominant arm after a 30 minutes of rest (Dinamap Compact T; Johnson & Johnson Medical Ltd, Newport, UK). In particular, BP was measured five times over 10 minutes and the mean of the last three determinations was considered for calibration. On the radial artery of the dominant arm, the pressure wave was recorded as the average of single waves consecutively obtained for eight seconds. Pressure wave determinations were considered reliable only if the variation of peak and bottom pressures of pressure waves was < 5%. The central pressure wave assessment was automatically derived from the radial measurements by a generalized transfer function [23]. Moreover, central waveforms were evaluated to identify the time to peak/shoulder of the first (T1) and second (T2) pressure wave elements during systole. The pressure at the peak/shoulder of T1 was classified as outgoing pressure wave height (P1), the pressure at the peak/shoulder of T2 was defined as the reflected pressure wave height (P2), either as an absolute value or as percent of ejection duration. Augmentation pressure (AP) was defined as difference between P2-P1, and augmentation index (AI) as [AP/pulse pressure (PP)] * 100. Aortic pulse wave velocity (PWV) was measured from carotid and femoral pressure waveforms. Carotid to femoral transit time (ΔT) was calculated from the foot-to-foot time delay between carotid and femoral waveforms. The distance between the landmarks of the sternal notch and femoral artery was considered to evaluate the path length between the carotid and femoral arteries (L), and PWV calculated as L/ΔT.

Statistical Analysis
Data are reported as mean and standard deviation or as absolute and percent frequency, as appropriate. Comparisons among more than two groups were performed by One Way ANOVA, followed by a Bonferroni post-hoc between-groups comparison. Chi-squared test was utilized for categorical variables. The association between continuous variables was assessed by Pearson product moment correlation coefficient (r) and P value. The relationship between gender and PWV was investigated by point biserial correlation coefficient and P value. As potential confounders for the relationship between ferritin and PWV we considered all variables listed in Table 1 and those associated with this biomarker with P ≤ 0.05 at univariate analyses (see last column in Table 1) were considered to be introduced into the multiple linear regression model. To avoid collinearity, we did not include iron and transferrin into the multiple liner regression model because these two variables lied into the same causal pathway between ferritin and PWV. Similarly, we did not adjust for insulin to avoid collinearity between this variable and the MATSUDA index. To assess the variance of PWV explained by each covariate into the model, we calculated the squared of the part correlation coefficient. The effect modification by ferritin on the relationship between hs-CRP and PWV was investigated by introducing into the same linear regression model ferritin (the effect modifier), hs-CRP and their interaction term (ferritin * hsCRP) as well as a series of potential confounders. The estimated increase of PWV associated to a fixed increase in CRP (+ 1 mg/l) across quartiles of ferritin (36 ng/ml, 86 ng/ml, and 159 ng/ml) was investigated by the standard linear combination method [24]. In multiple linear regression models, data were expressed as standardised regression coefficients (beta) and P value. All statistical analyses were performed using SPSS version 22 for Windows (Chicago, Illinois, USA) and STATA statistical package (version 13, Texas, USA).   (Table 1). Moreover, from the first to the third group there was a significant increase of hs-CRP (P < 0.0001), AST (P < 0.0001), ALT (P = 0.001) and γGT (P < 0.0001), while e-GFR was significantly decreased (P = 0.007). As expected, hypertensive IGT and T2DM patients showed a worse metabolic and inflammatory profile with a significant increase in BMI, hs-CRP, AST, ALT, γGT, fasting glucose and insulin and a significant decrease of the MATSUDA index when compared to those with NGT. No differences between IGT and T2DM patients were observed (Table 1). Furthermore, serum iron levels significantly decreased (P < 0.0001) and transferrin and TIBC significantly increased (P < 0.0001) from the first to the third group (P < 0.0001) ( Table 1). Also hemoglobin levels showed a significant reduction in T2DM group (P = 0.028). On the contrary, ferritin levels significantly increased from the first to the third group (P < 0.0001) ( Table 1). It's important to remark that hemoglobin and ferritin levels of all study population were within the normal range. In the post-hoc analysis both IGT (P = 0.002) and T2DM hypertensive patients (P < 0.0001) displayed significantly higher ferritin levels in comparison with HT/NGT patients.

Hemodynamic parameters
Peripheral and aortic hemodynamic parameters for the study population, according to glucose tolerance groups, are presented in Table 2. There were no significant differences among groups for peripheral BP values and heart rate. On the contrary, from the first to the third group of glucose tolerance, we noted a significant increase of central (c)-SBP (P = 0.002) and c-PP (P = 0.019), without significant difference for c-DBP (P = 0.855). Hypertensive T2DM patients showed a significantly higher c-SBP (P = 0.002) and c-PP (P = 0.023) values than HT/NGT, but similar to HT/IGT. Similarly, all indices of vascular stiffness progressively increased from the first to third group (P < 0.0001). In particular, PWV values were significantly higher in HT/IGT (P = 0.048) and HT/T2DM (P = 0.001) patients in comparison with HT/NGT, but without any significant differences between these two groups. Similarly, also AI and AP values were significantly higher in HT/IGT and HT/T2DM patients than HT/NGT but substantially comparable between them ( Table 2).

Effect modification analysis
In Table 3 the effect modification by ferritin levels on the hs-CRP-PWV relationship was investigated by crude and adjusted analyses (see Methods, Statistical analysis for details). On crude terms, serum ferritin was a strong modifier of the relationship between hs-CRP and PWV (P for effect modification = 0.01 and Fig. 2). The effect modification by ferritin on the hs-CRP-PWV relationship remained significant also after data adjustment for potential confounders (P for effect modification = 0.004, Table 3 and Fig. 2). As shown in Fig. 2, the estimated increase in PWV value portended by a fixed increase in hs-CRP (+ 1 mg/l) was progressively higher across increasing values of serum ferritin.
These results indicate that high serum ferritin levels are not only a risk factor of arterial stifness, but also amplify the harmful effect of inflammation on PWV.

Discussion
In this study, in patients with a wide range of glucose tolerance (from NGT to newly diagnosed T2DM), we found that serum ferritin levels increase in parallel to the worsening of glycometabolic parameters (particularly insulin sensitivity), the raise of inflammation (as assessed by hs-CRP) and the reduction of serum iron and hemoglobin levels (Table 1). Furthermore, these observations go in close parallellism with the increase of central hemodynamic parameters and arterial stiffness indicators, in particular carotid-femoral PWV, across the same groups (from NGT to T2DM). In our study, in a multiple linear regression model, ferritin resulted to be the strongest determinant of PWV explaining a 14.9% of the variability of this biomarker and the ferritin-PWV relationship remained significant also after data adjustment for well-known modulators of arterial stiffness such as insulin sensitivity, hs-CRP and renal function. Age was not significantly associated with PWV, thus it did not represent a confounder in the ferritin-arterial stiffness link. Another novel hypothesis tested by us, was that serum ferritin could act as an effect modifier on the relationship between inflammation and arterial stiffness in the same patient-population. According to this hypothesis, we found that the estimated increase in PWV value promoted by 1 mg/l increase in hs-CRP was significantly more consistent across increasing values of serum ferritin. Thus, the present results suggest that serum ferritin levels are not only a risk factor of arterial stiffness but also an amplifier of the damage induced by inflammation on PWV.
The association between serum ferritin levels and arterial stiffness is in line with the hypothesis that elevated body iron stores are associated with the atherosclerosis process development and in keeping with previous studies reporting an association between higher serum ferritin levels with arterial hypertension and metabolic disorders incidence [5][6][7][8][9]. Moreover, a strong association between serum ferritin levels and atherosclerotic injury has been reported in different settings of patients [25,26]. In particular, increased common carotid artery intima-media thickness and presence of carotid plaques has been associated with serum ferritin in haemodialysis patients [25] and patients with non-alcoholic fatty liver disease [26]. However, there is growing evidence demonstrating a strong association between higher serum ferritin levels and functional vascular damage. According with this, Ha et al reported, in a large cohort of healthy subjects, an association between ferritin and brachial-ankle PWV [18]. Moreover in haemodialysis patients, serum ferritin was associated with progressive arterial stiffness increase during a three years follow-up, but only for serum values > 500 ng/ml (19).
In hypertensive patients, only few data are reported in literature. In this regard, Valenti and coauthors [16] showed that hyperferritinemia (i.e. higher than 240 ng/ml in females and 320 ng/ml in males) was associated with high aortic stiffness measured as carotid-femoral PWV in patients with well-controlled hypertensives but with several comorbidities and already receiving drug treatment whereas data about the glycometabolic state were not available in this study. All these factors may explain why hs-CRP was not retained as an independent risk factor of PWV in the multiple regression analysis [16]. In respect to the study by Valenti et al, our study has the advantage to include younger naive hypertensive, without chronic conditions (in particular renal and liver diseases) and who received no pharmacological treatment thus avoiding/minimizing possible confounding factors.
Moreover, all patients were evaluated by OGTT to define glucose tolerance and the effect of insulin sensitivity on vascular damage was assessed by Matsuda index, a strong surrogate of euglycemic clamp. Finally, the most important observation was that, in our study, serum ferritin levels were within the normal range value.
It's plausible that in chronic conditions characterized by subclinical inflammation, such as arterial hypertension and T2DM, dysregulation of iron metabolism may lead to increased oxidative stress promoting arterial stiffening. According with this, exprimental and clinical studies demonstrated that increased ROS production during chronic iron overload plays an important role in the CV damage [1,27,28]. In fact, chronic iron overload can induce structural and functional changes in resistance arteries by reducing nitric oxide (NO) bioavailability resulting in an increase of O 2 − production through NADPH oxidase activity. These changes may involve local angiotensin 2 expression and ERK1/2 pathways, which may interfere with vascular remodelling and collagen deposition, thus favouring increased vascular stiffness [27]. Moreover, the same mechanisms facilitating endothelial dysfunction may also justify the increased vascular reactivity during chronic iron overload [1]. To prove this, iron restriction was able to attenuate vascular fibrosis and renal damage in renovascular hypertensive rats [2].
These pathophysiological aspects may strengthen the association between ferritin levels, an important biomarker of iron store, and arterial stiffness in patients with CV risk factors characterized by subclinical inflammation. In keeping with this observations, hs-CRP levels represent a significant predictor of PWV in the present study. Moreover serum ferritin acts as an effect modifier on the relationship between hs-CRP and arterial stiffness in the same population.
It is well known that serum ferritin is an acute-phase reactant, reflecting the degree of acute and chronic inflammation in several systemic diseases. However, emerging evidence suggests a potential causative role for ferritin in the inflammatory process [4]. At this regard, in vitro studies showed that ferritin may work as a local cytokine, activating Mitogen-Activated Protein Kinases (MAPK)-induced NF-kB in an iron-independent manner. This activity induces the increased expression of multiple proinflammatory mediators and a near 100-fold increase in inducible NO synthase that amplify oxidative stress [29,30]. In addition, ferritin is also able to directly modulate the lymphocyte function thus acting as pathogenetic player in the innate immune response [31]. Considering that our study population presented a normal inflammation and iron status, it is clinically plausible that ferritin per se might affect the increase in oxidative stress promoting those mechanisms that lead to vascular remodeling and increased arterial stiffness. Clinical practice supports this hypothesis, because ferritin represents a biomarker of disease progress and an independent predictor of various clinical outcome in different settings of patients [32][33][34]. Since the increase in carotid femoral PWV is associated with a significant risk increase for CV morbidity and mortality, we can speculate that the association between serum ferritin levels and PWV may be responsible for the negative prognostic effect of ferritin.

Conclusion
In conclusion, serum ferritin levels, within the normal range, represent an independent predictor of arterial stiffness in a large cohort of naïve hypertensive patients with different glucose tolerance and a strong effect modifier on the relationship between inflammation and PWV, thus a possible candidate for early marker of atherosclerosis at least in this setting of patients. This may be clinically relevant because ferritin is an easily determined laboratory parameters and widely used in medical practice.
The main limitation of the study is its cross-sectional design, therefore no causal link can be assessed.
In addition the measurements of hepcidin, a well-known regulator of body iron fluxes, were not available. This may be important because the determination of hepcidin levels could give more information about iron metabolism, also in subclinical inflammation. However, we evaluated transferrin saturation which is an important determinant of hepcidin release.

Ethics approval and consent to partecipate
The study protocol was approved by the The local Ethical Committee ( Comitato Etico Azienda Ospedaliera "Mater Domini") and conducted in accordance with the Declaration of Helsinki. Figure 1 Explained variance of pulse wave velocity (PWV), expressed in %, attributable to each covariate into the multiple model. Data are derived from  Effect modification by ferritin levels (fixed at predefined levels of this biomarker) on the relationship between high sensitivity C reactive protein (hs-CRP; + 1 mg/l increase) and

Figures
pulse wave velocity (PWV). Data are crude and adjusted (Table 3-Model with effect modification) estimates. See Methods-Statistical Analysis for more details.