Even in the healthy population, the heart ages together with other body organs. Aging may be considered a modern pandemic associated with a serious social and economic impact. The progressive increase in life expectancy is associated with higher prevalence of chronic age-related disease. In this light, understanding the mechanisms underlying this process and the physiological changes that occur with time, is crucial in improving the quality of life of the elderly and reducing the burden of age-related diseases. The aging process undeniably affects the cardiovascular system, and the prevalence of cardiovascular diseases increases over time (1). The aging process induces structural and functional changes such as vascular stiffening, myocyte hypertrophy and increased wall thickness, increased myocardial fibrosis, and extracellular matrix remodeling, which taken together lead to diastolic dysfunction characterized by reduced active filling of the left ventricle. Progressive left ventricular diastolic dysfunction is important, even in cases when the left ventricular mass is not remarkably increased during aging. However, the estimation of diastolic dysfunction could represent a marker for cardiac aging (1). Clinical factors which could accelerate the process of cardiac aging include: visceral thickness (obesity), diabetes mellitus, dyslipidemia, and hypertension. At the molecular level, it is believed that in cardiac myocytes – the reactive oxygen species, the transforming growth factor-beta, mitochondrial function – are associated with cardiac aging (2). Furthermore, the aging-related left ventricular diastolic dysfunction was showed to be one of the main risk factors for developing heart failure, which is most frequently heart failure with preserved ejection fraction (HFpEF). The most important risk factors for its development include: age, hypertension, metabolic syndrome, diabetes mellitus, renal dysfunction, and physical inactivity (2, 3). There is a wealth of information in the literature on the role of inflammatory cells and pathways during the acute injury and the regeneration processes, which are activating subsequently. Unfortunately, relatively little is known about the reasons which lead to chronic inflammation in heart failure as a sum of the initial inflammatory response, which represents only a trajectory for disease progression (4).

Inflammation is a complex defense mechanism in which leucocytes migrate from vasculature into damaged tissues to destroy the agents that can potentially cause tissue injury. Acute inflammation has a limited beneficial response, particularly during infectious challenge, whereas chronic inflammation is a persistent phenomenon that can lead to tissue damage. One hallmark of acute inflammation is that the leucocyte infiltrate is initially mostly neutrophilic, but monocyte cells predominate after 24 to 48 hours. In contrast, chronic inflammation is histologically associated with the presence of mononuclear cells such as macrophages and lymphocytes. (4) Although several explanations have been suggested, the mechanisms that control the transition from neutrophil to monocyte recruitment during the transformation from acute to chronic inflammation are poorly understood. It is possible that the interleukin-6 (IL-6)/soluble IL-6 receptor α (sIL-6Rα) complex plays an important role in this transition. IL-6 has a dual effect; at some levels it acts as a defending mechanism but in chronic inflammation it is rather proinflammatory. (4, 5) Immuno-senescence is an integral part of human aging that results in a decrease in the number of naive T and B lymphocytes, the accumulation of memory and effect of T and B cells, the production of defective antibodies, an increase in the production of autoantibodies, and in chronic low-grade inflammation. (4, 5) Among its most important features are slight elevations of the concentrations of pro-inflammatory cytokines, chemokines, and adipokines, such as interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1. (4) Pro-inflammatory cytokines stimulate the synthesis of C-reactive protein (CRP) in the liver, the level of which has been shown to increase in elderly individuals. The mechanisms for CRP elevation in patients with heart failure have not been completely defined (6). Possible theories include organ-congestion and hypoperfusion which causes secretion of IL-6 from hepatic, renal, endothelial, mononuclear, and, of course, cardiac myocytes. IL-6 causes CRP production in liver. This increased production could reflect a phenomenon of inflammatory state which causes development of diastolic dysfunction. While clinical signs and symptoms of inflammation are minimal or absent, this condition contributes to various molecular pathologies, leading to vascular damage and insulin resistance and, therefore, increases the risk of developing type 2 diabetes, cardiovascular disease, stroke, cancer, sarcopenia, neurodegeneration, and frailty. Furthermore, low-grade inflammation predicts mortality in elderly individuals who are affected by various pathologies. (4, 6)

A number of links have been proposed between inflammation and deterioration of health at older ages. IL-6 and CRP are among the most commonly used indicators of inflammation. (5, 6) Even in absence of chronic conditions, circulating inflammatory factors, such as interleukin IL-6, tumor necrosis factor (TNF)-α and soluble TNF receptor-1 (TNFR-1), and C-reactive protein (CRP) are usually two to four times higher in the elderly compared to young subjects. CRP is synthesized in the liver, and high-sensitivity CRP (hs-CRP) is a sensitive biomarker of systemic inflammation. Plasma levels of the inflammatory biomarker hs-CRP predict vascular risk with an effect estimate as large as that of total or high-density lipoprotein cholesterol. (5, 7)

The aims of this article were:

Study design. This study examined elderly people aged >65 years, 60 of whom had signs of HFpEF and comorbid diseases, while 18 comprised the healthy control group with no comorbidities. Of the most common comorbid diseases, the following were investigated: arterial hypertension, diabetes mellitus, obesity, and chronic renal dysfunction. Patients with verified chronic coronary arterial diseases, chronic obstructive pulmonary disease, progressive anemia, acute and chronic inflammatory diseases, malignant diseases, and also patients with progressive neurologic diseases and dementia as well as patients being on long-term anti-inflammatory therapy, and patients in whom the values of hs-CRP was >10mg/L were excluded.

Basic investigations. All examinees responded to previously prepared questionnaires addressing their medical history, presence of comorbidities, smoking, medication use, and Minnesota Living With Heart Failure Questionnaire, partly adjusted for the age group above 65 years. Measurements of body weight and height, measurement of the body mass index (BMI), measurement of blood pressure, cardiac frequency, 12-channel ECG, and taking blood for laboratory analyses were performed at the Gerontology Institute “13 November”, Skopje.

Inflammatory mediators. hs-CRP was analyzed with the following method: Turbidimetry, Integra 400 Rochе) (mg/L) from samples of venous peripheral blood. IL-6–was tested using the ECLIA (pg/mL) method from samples of venous peripheral blood. The investigation of these mediators was performed in the “Adrialab” (Sinlab) private biochemical laboratory.

Echocardiography and color Doppler of carotid blood vessels. All patients underwent two-dimensional color Doppler echocardiography and color Doppler of carotid blood vessels with SonoSite MicroMaxx (USA), with 2.5-MHz probes for all examinations. All echocardiographic measures were performed according to the Recommendations for Cardiac Chamber Quantification by Echocardiography in Adults: An Update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging 2015 (8) and Recommendation for the Evaluation of Left Ventricular Diastolic Function by Echocardiography: an Update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging 2016 (9). Gradation was performed according to several already established criteria: 1^st degree – postponed relaxation, 2^nd degree – pseudo-normalization, and 3^rd degree – restrictive type (9).

The level of the heart failure was estimated using the functional New York Classification for Hearth Failure (NYHA) according to the patients’ data given in their questionnaires. The patients in the groups were investigated according to NYHA (II-IV).

Carotid score. score 0: no plaques and thickness of intima-media complex (IMT) < 1 mm; score 1: thickened (≥1 mm) IMT; score 2: non-stenotic plaque (with or without IMT thickening); and score 3: stenotic (≥50% stenosis) plaque. Determination of the pick-systolic velocity and end-diastolic velocity (PSV/EDV) of the associated carotid artery (ACC).

In our investigated group of patients older than 65 years with clinical and echocardiographic diagnosis of HFpEF, it was found that a minority of patients had only one comorbidity 6 (12.2%), 18 (36.7%) had two comorbidities, and the same number of patients had three comorbidities, while 4 comorbid states were registered in 7 (14.3%) patients (Figure 1). The number of comorbid conditions did not depend significantly on the patients’ sex (Fisher exact, two tailed, p=0.53).

Regarding the type of the comorbidities, all patients from the investigated group had arterial hypertension, 36 (75.0%) female and 13 (26.5%) male patients.

Diabetes was found in 34 (69.4%) patients, non-significantly more frequently in the male patients – 10 (76.9%) vs 24 (66.67%) (Fisher exact, two tailed p=0.7). Dyslipidemia was not registered in this group of patients. Chronic renal insufficiency was found in 24 (49.0%) patients, non-significantly more frequently in the male patients – 7 (53.9%) vs 17 (47.2%) (Chi-square p=0.68). Sixteen patients (32.7%) were obese, non-significantly more frequently the women – 12 (33.3%) vs 4 (30.8%) (Fisher exact, two tailed p=1.0). Basic major cardiovascular characteristics of the investigated group are shown in Table 1.

The hs-CRP values significantly positively correlated with the number of the comorbidities of the investigated group (Spearman R=0.473; p=0.0006), i.e. the higher serum hs-CRP values were related to a greater (numerous) number of comorbidities, and vice-versa (Figure 2). In the group without comorbidities, 11 (36.7%) patients had hs-CRP values lower than 1 mg/L, while the other 19 (63.3%) had values ranging from 1 to 5 mg/L. There were no patients with comorbidities and hs-CRP values lower than 1 mg/L. The serum hs-CRP presented values higher than 5 mg/L in 2/6 patients with one comorbidity, 10 (55.6%) with two comorbidity, and in 21 (84.0%) with three and more comorbidities. The patients without comorbidities had significantly lower hs-CRP values in relation to the patients with two, three, and more comorbidities (p=0.00014, p=0.00015 respectively). Statistically significantly lower hs-CRP values were also registered in the group of patients with one comorbidity in relation to those with three and more comorbidities (p=0.0003).

The IL-6 value significantly positively correlated with the number of comorbidities in the investigated group (Spearman R=0.445; p=0.001), i.e. the higher serum interleukin values were associated with a greater number of comorbidities and vice-versa (Figure 3). In the group without comorbidities, all patients had IL-6 values lower than 5.5 pg/L, while the IL-6 values were higher than 10 pg/L in one patient with arterial hypertension as a comorbidity; in 7 (38.89%) patients with two comorbidities, and in 18 (72%) patients with three and more comorbidities. The patients without comorbidities had significantly lower interleukin values compared with the patients with three or more comorbidities (p=0.0001, p=0.0001 respectively). Significantly lower interleukin values were also registered in patients with one and two comorbidities (p=0.002; p=0.03 respectively).

Among patients of the investigated group with HFpEF, elevated LA volume index (>34 mL²) was found in 52 (86.6%) by echocardiography, which corresponded to the seriousness of diastolic dysfunction, as well as to the number and time duration of the comorbid diseases, while elevated LA volume index was found in 6 (10%) patients in the control group. Left ventricular hypertrophy was found in 55 (91.6%) of the patients, compared with the control group where a low level was found in only 2 (11.1%) patients. Among patients with the second level of diastolic dysfunction, being the most numerous, 46 (76%) had carotid score 1, 12 (20%) patients had carotid score 2, and 2 (3.3%) patients had carotid score 3.

The hs-CRP values and interleukin significantly positively correlated with the degree of the heart failure, analyzed with NYHA (Table 2). Elevated values of these parameters were found in patients with higher degree of heart failure, and vice-versa.

In the investigated group, hs-CRP non-significantly correlated with LV mass, LAVI max, and body mass index (BMI), and significantly negatively correlated with e’ parameter (R = -0.370; p=0.009). The hs-CRP serum values decreased with the elevation of the e’ parameter values and vice-versa. In the investigation group, IL-6 non-significantly correlated with e’, LV mass, LAVI mass, and BMI (Table 3).

In the control group, hs-CRP non-significantly correlated with e’, LV mass, and BMI, while interleukin non-significantly correlated with e’, LV, and BMI, and negatively correlated with LAVI max (R = -0.376; p=0.04). The serum interleukin values in the control group decreased with elevation of the LAVI max parameter, and vice-versa.

It is well-known that inflammation became one of the central fields of the investigation of the systolic heart failure over the last decade. There are also significant data which point to the role of inflammatory factors in development of diastolic dysfunction (10). It is now known that almost every nuclear cell type in the myocardium, including the heart myocytes, is capable of radiating pro-inflammatory cytokines as a response to various damage of the myocardium or stress, even in the absence of systemic immunologic activation (11, 12). The difference in pathophysiology between heart failure with a reduced ejection fraction (HFrEF) and HFpEF remains poorly understood, and effective treatment options are currently not available for HFpEF (13, 14). Therefore, a better understanding of the pathophysiology of HFpEF is required, which may eventually help to improve patient outcomes (15, 16). Numerous studies have concluded that the inflammation markers are more elevated in HFpEF, while the markers of cardiac stretch were more elevated in HFrEF (17). It is well documented that chronic inflammation is associated with aging-related morbidity in affected individuals. Therefore, the present study evaluated a group of seniors to determine whether results of a single measurement of the levels of two pro-inflammatory factors, IL-6 and hs-CRP, were associated with clinical and echocardiography parameters and whether they were predictors of HFpEF.

Patients with HFpEF are commonly older, and the majority have a history of arterial hypertension and frequently multiple comorbidities including diabetes, obesity, anemia, renal dysfunction, chronic obstructive pulmonary disease, etc. (2, 3). Each of these comorbidities has an impact on ventricular and vascular structure and function, and it is thus important to consider if HFpEF is a separate disease which requires specific therapy or whether it is simply an effect of cumulative comorbidities which follow the older population (3). Our study demonstrated that, compared with a relatively healthy group of elderly people, HFpEF patients have abnormalities in cardiac and vascular structure. Compared with the heathy control group, these patients had greater cardiac remodeling (concentric left-ventricular hypertrophy and left-atrial dilatation), as well as diastolic dysfunction (elevated E/e’, lower e’), and abnormal vascular function (IMT thickness, non-stenotic and stenotic plaques of carotid blood vessels). These comorbidities are associated with a unique clinical, structural, functional, and prognostic profile.

In our study, the serum level of IL-6 and hs-CRP showed significant association with the parameters of diastolic dysfunction, as well as with the level of left-ventricular hypertrophy and the parameters of the left atrial remodeling. This fact provides clinically significant information on the systemic immune abnormalities in patients with diastolic dysfunction. However, for hs-CRP values <3, there was no significant difference between the control and the investigated group, with only one comorbidity (arterial hypertension). This indicates that the progression for the patients >65 years with diastolic dysfunction into symptomatic HFpEF could be caused by inflammation and oxidative stress, especially since the majority of the elderly have associated diseases for which the systemic inflammatory nature has been demonstrated. Carotid score was is also in positive correlation with the level of diastolic dysfunction, which points to the role of atherosclerosis of the major blood vessels in occurring HFpEF. Hypertension and diabetes are the greatest possible precursors for HFpEF, and the progression from asymptomatic into symptomatic diastolic dysfunction is due, to a great extent, to development of left atrial dilatation and dysfunction, as well as to progression of left-ventricular hypertrophy. Our study showed positive correlations with parameters of left atrial remodeling, which corroborates that inflammation, through the left atrial remodeling, leads to progression from asymptomatic atrial dilatation, which is characteristic for the process of aging in HFpEF. The level of diastolic dysfunction significantly correlated with the level of carotid atheromatosis, the level of the inflammatory mediators in blood, and the level of the heart failure. The value of early mitral motion in mitral annulus, measured on the lateral side (e’), and which directly reflects diastolic function, falls with old age, which points to a decrease of the left-ventricular relaxation. In this study, it was in direct correlation with the number and the time-duration of comorbidities, which pointed to their impact on the development of diastolic function and development of HFpEF. Patients with lower e’ also had a higher level of inflammatory mediators in the blood, which indicates the impact of systemic inflammation in decreasing the left-ventricular relaxation (17, 18).

The limitations of our study are that we were not able to investigate more inflammatory mediators – TNF-alpha, IL-18, etc.

In the present study, hs-CRP was correlated with the parameters of carotid atheromatosis (IMT thickening, plaques presence), but there was also a correlation with the parameter of diastolic dysfunction in the healthy control group. It indicates that hs-CRP, besides being a verified marker for total cardiovascular risk, also helps perform a risk stratification of patients with diastolic dysfunction who still do not have symptoms of HFpEF in order to predict occurrence of HFpEF and to adequately decide an individual therapeutic program for each patient. Furthermore, randomized trial data addressing hs-CRP have been central to understanding the anti-inflammatory effects of statin therapy and have consistently demonstrated on-treatment hs-CRP levels to be as powerful a predictor of residual cardiovascular risk as on-treatment levels of low-density lipoprotein cholesterol (18). However, although hs-CRP is clinically useful as a biomarker for risk prediction, most mechanistic studies suggest that CRP itself is unlikely to be a target for intervention. If CRP is a downstream biomarker for atheromatosis, what do comparable data for the upstream “secondary messenger” cytokine IL-6 show? First, like hs-CRP, IL-6 levels measured in apparently healthy populations also predict future vascular risk; this observation was initially made in men in 2000, confirmed in women, and subsequently reproduced in more than 25 prospective epidemiologic cohorts worldwide (19, 20). IL-6 directly participates in conversion of cardiac myocytes into fibroblasts, and because of that the changes in IL-6 concentrations are directly related with cardiac dysfunction and changes of the cardiovascular matrix. Therefore, IL-6 is frequently discussed as a “remodeling” biomarker and also as independent from the other inflammatory markers, as a risk-factor for unwanted cardiovascular events and development of heart failure (20, 21). Moving upstream in the inflammatory cascade from CRP to interleukin IL-6 to IL-1 provides novel therapeutic opportunities for atheroprotection that focus on the central IL-6 signaling system and ultimately on inhibition of IL-1β-production.

Our results corroborate that IL-6, a cytokine which is a key mediator in many inflammatory, allergic, and infective diseases, is also significant in the states of chronic inflammation, while its correlation with the level of diastolic dysfunction and vascular atheromatosis could influence progression of this chronic inflammation into myocardial fibrosis that has been demonstrated in animal studies.

In this study, we showed that patients with diastolic dysfunction have elevated inflammatory mediators, which correspond to the seriousness of the disease, but whether these parameters have prognostic implications is the subject of many other studies. Existing results indicate that only the elevated hs-CRP values can be used as a screening risk factor in the senior “healthy population” for heart failure and other cardiovascular events (16). Diagnosis of heart failure without symptoms is a basic way to improve the therapy efficacy. Determination of specific, sensitive biomarkers, which reflect the complex pathophysiology of heart failure, could also be used as a significant parameter for screening patients with heart failure, who need further extensive diagnostic examinations (4, 9). Generally, clinical studies in which efforts are made to modulate the inflammatory processes in patients with heart failure have to a great extent been disappointing, except for some smaller subgroups with an extensive anti-inflammatory approach. This does not have to mean the end of the era of cytokine, but only indicates the challenges of this therapeutic approach for modulating the cytokine net, which represent the useful as well as the harmful effects on myocardial remodeling.

It is quite probable than HFpEF represents a unique synergistic interaction between the effect of the aging process itself, hypertension and comorbidities, primarily cardiovascular comorbidities, which cause heart and vascular remodeling and dysfunction in absence of other cardiac diseases, which lead to decreased pump power and ejection fraction (13, 14, 22). Up to now, prevention for HFpEF could only be effective by treatment of the risk factors. Discovering novel multidimensional strategies which would improve endothelial dysfunction and heart remodeling poses a challenge. Novel, randomized strategies are needed for discovering evidence-based treatment for HFEF (13). Connecting discoveries in cardiology with an aspect of the aging process and comorbidities in geriatric cardiology would have larger significance in discovering novel modalities in the treatment (23, 24).

While several attempts to target this condition using therapeutic anti-inflammatory regimens to reduce cardiovascular mortality have failed, targeting the whole CRP/IL-6/IL-1 axis with monoclonal antibodies could open new therapeutic lines in cardiovascular pathology and offer novel insights into the aging process of the heart (22-24).