Association Between Timp-3/Angiotensin II, Profile and Cardiac Remodeling in Patients with Essential Hypertension and Heart Failure with Mid- Range Ejection Fraction

Introduction Traditionally cardiac extracellular matrix (ECM) is thought to be a relatively inactive structure, which takes the role as scaffolding for cardiac myocytes and vessels. Recent studies show evidence that cardiac ECM is metabolically active and dynamic structure. Changes in cardiac ECM turnover are suspected of contributing to the genesis and progression of heart failure [1]. Degradation Abstract


Introduction
Traditionally cardiac extracellular matrix (ECM) is thought to be a relatively inactive structure, which takes the role as scaffolding for cardiac myocytes and vessels. Recent studies show evidence that cardiac ECM is metabolically active and dynamic structure. Changes in cardiac ECM turnover are suspected of contributing to the genesis and progression of heart failure [1]. Degradation of ECM's structural proteins occurs through the action of matrix metalloproteinases (MMPs), which are regulated by tissue inhibitor of metalloproteinases (TIMPs). TIMP-3 is the only TIMP that is ECM-bound and could exert tissue-specific effects [2,3]. However, the exact role of TIMP3 in hypertension remains to be understood. The detection of subclinical left ventricular hypertrophy (LVH) is made difficult by several factors. "Firstly, the clinical disease processes do not cause signs and symptoms; consequently, LV remodeling may remain an unrecognized and insidious process for a prolonged period of time. Secondly, LVH is not easily detectable using standard clinical means such as a patient's history, physical exam or electrocardiography (ECG). Specialized testing tools are usually needed for that subspecialty expertise to perform and interpret" [4].
There is growing evidence that supports the notion that angiotensin II (AII) may directly cause cardiovascular and renal diseases, independent of its hypertensive effect. Thus, recent in vivo work, coupled with in vitro findings, has provided new insights into the molecular and cellular mechanisms of AII-mediated cardiovascular and renal diseases [5]. Angiotensin II exerts direct effects on vascular remodeling and function [6]. TIMP-3 could be implicated in the progression of vascular diseases, independently from its ability to inhibit MMPs. In this regard, in vitro and in vivo adenoviral overexpression of TIMP-3, but not TIMP-1 or -2, has shown to promote apoptosis in vascular smooth muscle cells. In vivo experiments have shown that this effect was not achieved when synthetic MMP inhibitor was used [7,8]. Arterial hypertension (AH) is a leading cause for large number of heart failure (HF) cases. Heart failure with mid-range ejection fraction (HFmrEF) is a syndrome, defined by: (1)-Left ventricular ejection fraction (LVEF%)-40-49%; (2)-Symptoms and/or signs of heart failure; (3a)-Elevated levels of natriuretic peptides; (3b)-At least one additional criterion: relevant structural heart disease (left ventricular hypertrophy/left atrial enlargement) or diastolic dysfunction-2016 European Society of Cardiology (ESC) Guidelines for the diagnosis and treatment of acute and chronic heart failure [9].

Clinical draft
All patients were residing in the vicinity of the Pleven University Hospital. Subjects' sera were taken from October 2016 to May 2017. All the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, as revised in 2000. Approval of local Ethics Committee was obtained and informed consent from adult research participants was obtained too. The study group consisted of 56 patients with AH and HFmrEF, mean age 65.62±9.69. Forty-one of patients were with hypertension mediated organ damage (HMOD) and 15 were without. Patients were divided in two subgroups-subjects with mid-range ejection fraction and left ventricular hypertrophy (n=32), mean age 62.5±12.58 years (HFmrEF+LVH); subjects with mid-range ejection fraction without left ventricular hypertrophy (n=24), mean age 60.4±8.4 years (HFmrEF-LVH) ( Table 1). These values were compared to serum TIMP-3/Angiotensin II profile in 22 age and sex matched controls with no family history of diabetes, atherosclerosis or hypertension, mean age 56.4±5.53 years. Hypertension-Mediated Organ Damage Assessment: All patients were examined for HMOD via anamnesis (for established cardiovascular or premature CVD, renal, cerebrovascular or ophthalmological damage) and physical examination and pulse pressure assessment (in older people) >_60mmHg.

Instrumental tests
a.
Arterial blood pressure was measured using a standard anearoid sphygmomanometer, to the nearest 2 mmHg, in the dominant arm after at least 10-min rest in supine position.

b.
ECG was performed for LVH assessment (Sokolow-Lyon index >35 mm, or R in aVL >_11 mm; Cornell voltage duration product >2440mm.ms, or Cornell voltage >28mm in men or >20mm in women).

Elisa
Enzyme-linked immunosorbent assay (ELISA) was used for measuring AII and TIMP-3 levels. TIMP-3 levels were measured in serum samples using enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, DY973) according to the manufacturer's instructions. Angiotensin II levels were measured in serum samples using enzyme-linked immunosorbent assay ALPCO Diagnostics (Salem, MA, USA, 74-ANGHU-E01) according to the manufacturer's instructions.

Statistical analyses
The research data was processed with the computer programs EXCEL (Microsoft Corporation, Redmond, WA) and STATGRAPHICS plus (Manugistics, Rockville, MD) for WINDOWS. All results were described in tables, graphs, numerical values (mean ± SD, share indicators and correlations). For assessment and conclusions in the case of normal distribution the Student t-test, Fisher's F-test (ANOVA) and post-hoc tests (LSD, Tukey HSD, Scheffe, Bonferroni, Newman-Keuls, and Duncan) were used, and for distribution, different from the normal -the K-W (Kruskal-Wallis)-test. The level of significance was determined as p < 0.05. In cases with different from normal distribution, median was used (M), together with first and third quartile Q1 and Q3; (twenty-fifth and seventy-fifth percentile P25 and 75P).

Discussion
The conceptual role for AII, as a local mediator of fibrosis, is suggested by studies in which circulating AII is chronically increased from either endogenous or exogenous sources [11]. Mounting evidence points towards an authentic signalling capacity for TIMPs distinct from their MMP-inhibitory activity. It also seems to play an important role in the regulation of apoptosis, cell survival, growth, migration, differentiation, angiogenesis, inflammation and overall ECM remodelling. Due to these mechanisms TIMPs could play a vital role in the process of cardiac remodelling [12,13]. A suggestion about a link between TIMPs and the renin-angiotensin-system is firstly given by Kang [14]. They identified human angiotensin-II-type-2-receptor as a novel TIMP-3 interacting partner, linking TIMP-3 with the renin-angiotensin system. Although, the pathophysiological roles and signaling mechanisms of angiotensin-IItype-2-receptor are still largely unknown, but they were shown to be increased during hypertrophy and ischemic heart disease. Our results show decreased TIMP-3 levels and increased AII in all patients. Interestingly, the TIMP-3/AII profile is different according to presence of LVH. This can be described by the next findings: TIMP-3 levels in patients with HFmrEF-LVH are statistically significantly higher than those in HFmrEF+LVH. On the contrary-AII levels in HFmrEF-LVH are statistically significantly lower than these in HFmrEF+LVH.
These results can be summarized with the next two ratios: i.
Profile of patients with HFmrEF+LVH-decreased TIMP-3/ increased AII Our results are consistent with authors [15] who found that recovery of TIMP-3 content in the failing myocardium is associated with reversing cardiac remodeling, highlighting the influence of this critical matrix constituent in maintaining normal cardiac structure and function. We report the existence of an association between changes in levels of TIMP-3/Angiotensin II profile and cardiac remodeling. Determination of serum TIMP-3/Angiotensin II profile may be a useful method for monitoring of development and progression of LVH in patients with essential hypertension and heart failure with mid-range ejection fraction. This is a pilot study. Although these findings should be confirmed in a larger study, our data suggest that changes in the TIMP-3/AII balance may play an important role in the structural, functional, and clinical manifestations of cardiac remodeling. However further examination and prospective studies are needed to clarify the MMP-independent biological functions of TIMP-3 and fully understand its effects and exact relevance on contributing to the cardiac remodeling.