Relationship between skin blood flow regulation mechanisms and vascular endothelial growth factor in patients with metabolic syndrome

Abstract

The focus of this paper is the determination of endothelial dysfunction in patients with metabolic syndrome (MetS) and the establishment of a relationship between the traditional biomarkers of endothelial dysfunction and the vascular tone regulation indices obtained from indirect cold tests in MetS patients. Our investigation was conducted on 30 patients aged 45.5±9 years. The control group comprised 14 healthy subjects aged 48.2±2.4 years. The mechanism of vascular tone regulation was investigated using the wavelet analysis of skin temperature oscillations (WAST). The degrees of microvascular vasoconstriction and vasodilatation were determined during contralateral cold tests in the endothelial (0.02–0.0095 Hz), neurogenic (0.05–0.02 Hz) and myogenic (0.05–0.14 Hz) frequency ranges. In MetS patients, vasoconstriction indices were higher and vasodilatation indices were lower than in the subjects of the control group, which is indicative of disorders in the mechanisms of microvascular tone regulation. These indices correlate with the metabolic parameters and VEGF (vascular endothelial growth factor) levels. The correlation of vasoconstriction and vasodilatation indices with the main factors of the metabolic syndrome testifies that the biological and functional aspects of the endothelial dysfunction are closely related.

Keywords

Endothelial dysfunction wavelet analysis of skin temperature oscillations metabolic syndrome

1 Introduction

Endothelial dysfunction is an early event in the development and progression of a large number of cardiovascular (CV) diseases [1, 2]. Various human studies have shown that the measurement of endothelial dysfunction may offer prognostic information on vascular events. At present, there is substantial evidence in support of the view that destruction of the endothelial structure and function can be considered as an essential component of metabolic syndrome (MetS) [3]. The most important signs of MetS are abdominal adiposity (waist circumference ≥94 cm for men and ≥80 cm for women) associated with two (or more) of the following factors: high triglyceride level (≥1.7 mmol/l), decreased high-density lipoprotein cholesterol (≤1.03 mmol/l for men and 1.29 mmol/l for women) or specific treatment of this type of dyslipidemia, arterial blood pressure increase (≥130/85 mm Hg) or antihypertensive treatment, high fasting glucose level (≥5,6 mmol/l) or previously diagnosed type 2 diabetes

The endothelial cells lining the internal walls of the circulatory system are the primary target for haemodynamic, biochemical and immune factors of the circulating blood. However, the barrier action is not the only function of the endothelium; it is a hormone-active tissue in its own right, and it produces a variety of biologically active substances. Endothelial dysfunction is the lesion of the whole blood-vascular system manifesting itself as a reduction of endothelium-dependent vasodilation and vascular remodelling, which is a precursory symptom of the pathological processes in the cardiovascular system [4, 5].

Vascular endothelial pathology can be diagnosed by using biochemical and functional markers. Among these biochemical markers are biologically active substances synthesized by the endothelial cells and expressed on their surface, such as von Willebrand factor, endothelin-1, adhesion molecules (E-selectin, P-selectin), plasminogen activator inhibitor-1, thrombomodulin and fibronectin [6]. One of the most recognized biomarkers of endothelial dysfunction is the vascular endothelial growth factor (VEGF), which contributes to the pathogenesis of atherosclerosis, hypertension and type 2 diabetes. The expression of cytokines increases in hypoxia, thereby providing epithelium regeneration and the formation of new vessels [7]. Indications of an increase in VEGF were found in MetS patients [8]. NO and thromboxane, being the most important mediators of vasoregulation are described in the majority of publications, however few of them consider the effects of VEGF on the vascular tone, which is a motive for studying this problem in our investigation [9].

Microalbuminuria (MAU) is also included among the well-established cardiovascular mortality predicators in patients with type 1 and type 2 diabetes and hypertension. MAU is related to insulin resistance, dyslipidemia and central obesity and can enter the metabolic syndrome. Many scientists regard MAU as a marker of endothelial dysfunction [10].

To date, the skin vessels have been considered an easily accessible and potentially representative vascular bed that can be used to study endothelial function [5, 11]. Thus, at present, the skin is used as a model to investigate vascular mechanisms in a diversity of disease states, including hypercholesterolemia [12], hypertension [13], renal disease [14], type 1 and 2 diabetes [15 –17], peripheral vascular disease [18], heart failure [19] and obesity [20].

The methods most generally used for studying skin blood flow are the optical methods, such as photoplethysmography and laser Doppler flowmetry (LDF) [21, 22]. Capillary microscopy serves as a valuable diagnostic tool in the study of cutaneous microcirculation [23]. The analysis of skin blood flow characteristics can be performed using high-resolution thermometry, because temperature oscillations on the skin surface (ST oscillations) are caused by changes in the skin vascular tone [24, 25]. The spectral wavelet analysis of the variable component of LDF and ST signals allows one to estimate blood vessel conditions, vascular tone and mechanisms responsible for blood flow regulation in the microcirculatory bed. The spectrum of skin blood oscillations is divided into five sub-ranges, corresponding to different factors of vascular tone regulation: pulse wave (0.5–2 Hz), breath wave (0.14-0.5 Hz), myogenic oscillations (0.05–0.14 Hz), neurogenic activity (0.02–0.05 Hz), and endothelial activity (0.0095–0.02 Hz [26]. A cross-spectral analysis of the variations in blood pressure waveforms and temperature shows a high degree of correlation between the spontaneous fluctuations of skin temperature and the vasomotor activity of small arteries and arterioles in subcutaneous tissues [27]. Weak phase coherence between temperature and blood flow is observed for unperturbed skin, and after heating it increased in all frequency intervals [28]. ST pulsations mirror the functional state of the microcirculation system and ST monitoring can be used to monitor microvessels tone control within the frequency ranges corresponding to endothelial, neurogenic and myogenic activities [29].

The oscillations of blood flow with a frequency of the order of 0.01 Hz are associated with endothelial activity, in particular, with the activity of nitric oxide (NO) synthesis. Studies employing data obtained after the iontophoresis of acetylcholine (an endothelium-dependent vasodilator) and sodium nitroprusside (an endothelium independent vasodilator) have confirmed this [30, 31]. It is common practice to estimate the state of the skin blood flow using simple functional tests, such as a cold pressor test and a local heating test [32, 33].

Today, the lack of a unified diagnostic criterion for endothelial dysfunction generates a need for the development and verification of new methods based, in particular, on the wavelet analysis of skin temperature (WAST) [34]. The purpose of our investigation is to find a relationship between the traditional biomarkers of endothelial dysfunction and the indices of vascular tone regulation obtained using the WAST technique during the contralateral cold pressor test in MetS patients.

2 Methods

The study group comprised 30 subjects aged 27–55 years, 22 of whom were males (73%). MetS was diagnosed according to the IDF (International Diabetes Federation) criteria (2005) [35]. The exclusion criteria were acute coronary syndromes, uncontrolled hypertension, heart failure, pregnancy, inflammatory disorders, or history of renal disease or other severe chronic diseases. The control group consisted of 14 apparently healthy subjects aged 48.2±2.4 years (7 males and 7 females).

Measurements related to the CV risk included resting arterial blood pressure; body mass index (BMI); waist circumference; lipid profile: total cholesterol (TC), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), and triglyceride (TG) levels; fasting and postprandial blood glucose; serum creatinine and insulin level. Insulin resistance was estimated using homeostasis model assessment (HOMA-IR) from fasting serum glucose and fasting serum insulin.

The level of vascular endothelial growth factor A (VEGF-A) in blood serum and the level of urinary microalbumin were measured using an electrochemiluminescence method. Albuminuria was defined as microalbuminuria (MAU) (urine albumin concentration 300 mg/g of creatinine in spot urine collected in the morning). The glomerular filtration rate (eGFR) was calculated with the formula CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration), ml/min/1.73 sq.m.

Participants were in a fasting state for at least 10 h before the study. No subjects were smokers, and they were also advised not to take antihypertensive or vasodilation drugs on the day of examination. Patients were examined in a quiet, temperature controlled room (20–25°C). All the procedures were approved by the Local Ethics Committee (the number of the approval from the ethical committee is 12). All the participants signed informed consent forms.

The local medical ethics committee of the Perm State Medical Academy approved the study. Each subject gave verbal and written consent prior to participation in the study.

2.1 Contralateral cold test

The cold pressor test (a natural constrictive test [32]), was used to evaluate vascular tone regulation mechanisms in the study of low-frequency fluctuations of skin temperature. During the cold test, the subject was in a supine position, and measurements were taken at a room temperature of 22.5±0.5°C. The skin temperature of the back surface of the distal phalanx of the index finger of the right hand was recorded with the device “Microtest” (Perm, Russia), providing a temperature resolution of 0.001°C. The left hand was immersed in a bath of an ice-water mixture (temperature 0°C) for 3 minutes. The ST was measured continuously: during the 10 minutes prior to the test (t1), during the cold test (t2) and then after the test for three (t3) and then for ten (t4) minutes.

The choice of the duration of the intervals t1, t2, t3, t4 depends on several factors. The interval t1 was taken as 10 minutes to provide statistically significant results in the endothelial range (about 10 oscillations). Immersion of subject’s hand into cold water maintained at zero degree C is a procedure with a high-stress level, and therefore the maximum cold exposure time during which subjects can stay in their comfort zone does not exceed 3 minutes. The choice of 3-minutes for the interval t3 was due to the fact that an increase in tissue temperature during the first three minutes triggers the axon reflex mechanism and only then the NO generation mechanism [36]. Hence, the dynamics of tissue responses within the first three minutes after cooling may differ from subsequent changes in the blood flow. The interval t4 was chosen as 10 minutes in order to obtain reliable statistics in the endothelial range (about 10 oscillations) after the termination of the cold test.

2.2 Software and statistical analysis

Using the continuous wavelet transform, the skin temperature function of one variable (time) can be represented as a 2D (time and scale) space [37, 38].

Thus, we have $W (a, b) = \frac{1}{a} \int_{- \infty}^{+ \infty} f (t) ψ^{*} (\frac{t - b}{a}) dt,$ (1) where ψ is the analyzing wavelet, t is the time, * is complex conjugation, b is the time shift (wavelet position), and a is the oscillation scale, which corresponds to the inverse of the frequency (ν): $ν = \frac{1}{a}$ (2) For this task wavelet transforms are made with respect to a Morlet mother wavelet. $ψ (t) = exp (2 π it) exp (- \frac{t^{2}}{κ^{2}})$ (3) When choosing parameters for the Morlet wavelet, care must be taken to ensure the balance between the time and frequency resolution. In this work, we restricted ourselves to a relatively short duration of cold test (180 s) and a maximum scale of pulsation (70 s –the mean period of oscillation, caused by endothelial activity). Morlet wavelet with κ= 1 provides a rather good time resolution for this case, but insufficient frequency resolution, which as a result leads to overlapping of the frequency ranges. To diminish this effect, the boundaries of the frequency ranges were corrected (in comparison with [26]) in the following way: myogenic frequency range 0.14-0.07 Hz, neurogenic –0.031-0.026 Hz, endothelial –0.0139-0.0095 Hz.

A two-dimensional wavelet transform yields a set of data (Fig. 1, left panel). This form of data representation is quite specific and is difficult to interpret. A more common and easy-to-understand representation can be obtained after the so-called inverse wavelet-transform [33].

Fig.1

Wavelet-plane (Y-axis indicates the scale of oscillations a = 1/ν, where ν denotes frequency and the X-axis indicates time) and the appropriate skin temperature oscillation amplitudes acquired during the contralateral cold test (600 ... 780 sec). Blue colors correspond to low values of wavelet coefficients, red colors - to large. Upper panel – ST oscillations in the endothelial, middle panel – in the neurogenic, and lower panel – in the myogenic frequency ranges.

$f (t) = \frac{1}{a^{2} C_{ψ}} \int_{0}^{+ \infty} \int_{- \infty}^{+ \infty} W (a, b) ψ (\frac{t - b}{a}) dadb$ (4) where C_ψ is given by $C_{ψ} = \frac{1}{2 π} \int_{- \infty}^{+ \infty} {| ω |}^{- 1} {| \int_{- \infty}^{+ \infty} ψ (t) e^{- i ω t} dt |}^{2} d ω$ (5)

Inverse wavelet allows us to distinguish oscillations in one region of a spectrum from the others (Fig. 1, right panel). Oscillation amplitudes provide us with the same information as the spectral characteristic of the signal on a wavelet plane.

The contributions of different vascular tone regulation mechanisms were assessed by selection of the value of the RMS (root-mean-square) amplitudes of the skin temperature oscillations < ΔT>within the corresponding frequency range. For each frequency range, we obtained an estimate of the change in the root-mean-square amplitudes of the skin temperature oscillations: ST_i/ST₁, where ST₁ are the root-mean-square amplitudes before the cold test and ST_i are the root-mean-square amplitudes for the corresponding time intervals (during the test, and within 3 and 10 minutes after the test).

The obtained values are denoted as the indices of vasoconstriction and vasodilation. For the indexing of the vasoconstriction index (IVC), we took k = ST₂/ST₁, where ST₂ are the oscillation amplitudes observed during the test. For the index of post-cold vasodilatation-1 (IPV-1), we took k = ST₃/ST₁, where ST₃ are the oscillation amplitudes within the first three minutes after the test. For the index of post-cold vasodilatation-2 (IPV-2), we took k = ST₄/ST₁, where ST₄ are the oscillation amplitudes within the interval from 3 to 10 minutes after the test.

The original algorithms of the wavelet analysis were realized in C++. The data are represented as M±SD, where M is the average value and SD is the standard deviation. A comparison between the groups was made using a non-parametric statistical test (the Mann–Whitney U-test). Investigation of interrelations between the quantitative attributes was carried out using the correlation-regression analysis techniques and was supplemented with a non-parametric method (Spearman’s rank correlation criterion), which made it possible to reduce the effect of incidental emission. The paired t-test was used to compare the paired data; p values < 0.05 were considered statistically significant. Statistical analysis was performed using a statistical software package called “Statistica 6.0”.

3 Results

Our study shows that the levels of cholesterol (TC), triglycerids (TG), low-density lipoproteins (LDL), glucose, uric acid insulin and eGFR were significantly higher in the MetS group compared with the control group (P < 0.05) (Table 1). The levels of both markers of endothelial dysfunction (MAU and VEGF) were substantially higher in the MetS group (P < 0.05) (Table 2).

Table 1
Main measured values in the MetS and control group

Parameters (units) MetS (n = 30) Control (n = 14) p

Age (years) 45.5±9,0 48.2±2.4 0.7

Male sex (%) 22 (73) 7 (50)

Waist circumference (cm) 113.4±12.5 80.7±11 0.0001

BMI (kg/m²) 37.5±4.4 27±1.3 0.0001

Systolic blood pressure (mmHg) 148.5±14.8 128.3±7.2 0.0001

Diastolic blood pressure (mmHg) 91.7±19.1 78.5±5.3 0.0001

Fasting glucose (mmol/L) 5.8±0.8 4.21±0.5 0.0001

Total cholesterol (mmol/L) 5.99±1.2 4±0.7 0.004

LDL cholesterol (mmol/L) 5.1±3.2 2.43±0.4 0.003

HDL cholesterol (mmol/L) 1.4±0.6 1.4±0.1 0.3

Triglycerides (mmol/L) 2.1±1.4 0.57±0.11 0.001

Creatinine (μmol/L) 75.8±12.5 66.0±10.1 0.2

Uric acid (mmol/L) 379.4±45.4 241.5±86.1 0.009

Insulin (μU/ml) 19.8±8.6 6.7±1.2 0.001

HOMA-IR (U) 5.2±2.3 1.25±0.7 0.01

eGFR (ml/min/1,73 sq.m.) 88.5±5.8 112.7±7.6 0.001

Parameters (units)	MetS (n = 30)	Control (n = 14)	p
Age (years)	45.5±9,0	48.2±2.4	0.7
Male sex (%)	22 (73)	7 (50)
Waist circumference (cm)	113.4±12.5	80.7±11	0.0001
BMI (kg/m²)	37.5±4.4	27±1.3	0.0001
Systolic blood pressure (mmHg)	148.5±14.8	128.3±7.2	0.0001
Diastolic blood pressure (mmHg)	91.7±19.1	78.5±5.3	0.0001
Fasting glucose (mmol/L)	5.8±0.8	4.21±0.5	0.0001
Total cholesterol (mmol/L)	5.99±1.2	4±0.7	0.004
LDL cholesterol (mmol/L)	5.1±3.2	2.43±0.4	0.003
HDL cholesterol (mmol/L)	1.4±0.6	1.4±0.1	0.3
Triglycerides (mmol/L)	2.1±1.4	0.57±0.11	0.001
Creatinine (μmol/L)	75.8±12.5	66.0±10.1	0.2
Uric acid (mmol/L)	379.4±45.4	241.5±86.1	0.009
Insulin (μU/ml)	19.8±8.6	6.7±1.2	0.001
HOMA-IR (U)	5.2±2.3	1.25±0.7	0.01
eGFR (ml/min/1,73 sq.m.)	88.5±5.8	112.7±7.6	0.001

Main measured values for the groups (M±SD).

Table 2

Level of serum VEGF and microalbumin

Values, unit of measurement	Median [25−75%]
	MetS (n = 30)	Control (n = 14)	P
VEGF, pg/ml	197 [58.2−288]	75 [0−96]	0.002
Microalbumin, mg/g	25.75 [11.7−34.0]	9.4 [5.3−13.2]	0.001

p – the significance of differences between the groups under comparison.

LDL cholesterol – low-density lipoproteins, HDL cholesterol – high-density lipoproteins, BMI – body mass index, eGFR – Estimated Glomerular Filtration Rate, BMI – body mass index.

We established a direct correlation between the value of the waist circumference (WC) and LDL (r = 0.5; p = 0.04). The level of insulin resistance (HOMA-IR) is associated with the level of uricemia (r = 0.48; p = 0.02). Other correlations between biochemical markers and endothelial dysfunction markers are given in Table 3.

Table 3

Correlations between biochemical markers and endothelial dysfunction markers

	Microalbumin	VEGF	Diastolic blood pressure
Waist circumference	NS	NS	0,5
eGFR	–0,6	–0,7	NS
BMI	NS	0,4	0,5
Triglyceride	0,4	0,7	0,5
Uric acid	NS	NS	0,4
Creatinine	NS	0,7	NS
HOMA-IR	NS	0,4	NS
Diastolic blood pressure	0,7	0,4	NS

eGFR – estimated glomerular filtration rate, BMI- body mass index, NS- non significant correlation.

During the cold test, the amplitude of the skin temperature oscillations in healthy subjects in the endothelial, neurogenic and myogenic frequency ranges exhibited a significant twofold decrease (Table 4). Upon completion of the cold test, an increase in temperature resulted in increasing temperature oscillation amplitudes and, practically, to their recovery to the initial values within the first 3 minutes.

Table 4

Mean values of skin temperature oscillation amplitudes (*10³, °C) in the endothelial, neurogenic and myogenic frequency ranges in the time intervals t₁ – t₄ for different groups of patients

Oscillation range	Group	ST₁ (t₁)	ST₂ (t₂)	ST₃ (t₃)	ST₄ (t₄)
Endothelial	Control	21.4±13.9	9.6±3.9	20.1±13.9	18.9±12.7
			p1-2 < 0.001	p1-2 = 0.016	p2-4 = 0.019
	MetS	23.9±11.8	13.5±7.5	20.4±18.1	15.7±10.7
			p1-2 < 0.001	p1-3 = 0.030	p1-4 < 0.001
				p2-3 = 0.016	p3-4 = 0.017
Neurogenic	Control	8.9±5.0	3.2±2.2	7.0±4.8	12.9±23.7
			p1-2 = 0.001	p2-3 = 0.009	p2-4 = 0.006
	MetS	9.2±6.1	7.5±10.6	9.5±7.2	6.8±5.4
			p1-2 = 0.016	p2-3 = 0.012	p1-4 = 0.002
					p3-4 = 0.002
Myogenic	Control	3.0±1.9	1.5±1.8	2.2±1.7	2.5±2.1
			p1-2 = 0.008	p1-3 = 0.03	p1-4 = 0.035
				p2-3 = 0.035	p2-4 = 0.019
	MetS	2.2±1.7	1.2±0.7	2.1±2.1	1.6±1.1
			p1-2 < 0.001	p2-3 = 0.011	p1-4 = 0.001
					p2-4 = 0.013

The response to cooling in MetS patients differed essentially from that in healthy people.

After the cold test, temperature oscillation amplitudes increased, yet they did not reach the initial value.

The indices of vasoconstriction and post-cold vasodilatation are summarized in Table 5. The index of vasoconstriction (IVC) shows that the amplitude of the temperature oscillations decreases compared to the initial values. It also indicates the vascular response to cooling. The indices of post-cold vasodilatation (IPV-1 and IPV-2) show that the amplitude of the temperature oscillations increases after the cold test, thus making it possible to describe the activity of the vasodilatation mechanisms.

Table 5

Values of the indices of vasoconstriction (IVC) and post-cold vasodilatation (IPV-1 and IPV-2)

Oscillation range	Group	IVC	IPV-1	IPV-2
Endothelial	Control	0.47±0.19	1.11±0.51	1.03±0.49
	MetS	0.62±0.18^*	0.71±0.29^*	0.64±0.26^*
Neurogenic	Control	0.43±0. 23	0.95±0.44	0.9±0.25
	MetS	0.95±0.92^*	0.83±0.37	0.71±0.34^*
Myogenic	Control	0.47±0.23	0.75±0.34	0.71±0.18
	MetS	0.61±0.32	0.84±0.27	0.77±0.29

^*p < 0.05 – the significance of differences between the groups in the appropriate frequency ranges.

Therefore, it can be suggested that an adequate response of the endothelium to cold stress is characterized by an approximately twofold reduction in the amplitude of oscillations, and this corresponds to the values of the vasoconstriction indices of 0.54±0.24. The values of indices of vasodilatation for the control group approach unity and lie in the interval 1.0±0.55.

The IVC values in the MetS group are much higher than those in healthy subjects, and this is indicative of less qualitative vasoconstriction. The values of the indices of post-cold vasodilatation (IPV-1 and IPV-2) are significantly lower, because the amplitudes of the skin temperature oscillations do not increase to the initial values. These phenomena can be attributed to a vasodilatation disorder. Significant vasodilatation disorders have been revealed in endothelial and neurogenic frequency ranges.

The value of IVC in the MetS group correlated with the insulin level (r = 0.42, p = 0.03; r = 0.64, p = 0.01) and MAU (r = 0.46, p = 0.018; r = 0.44, p = 0.03, Fig. 2, left) in the endothelial and the neurogenic ranges, respectively. The level of H_BA1_C increased as the IPV-2 decreased in the neurogenic (r = –0.87; p = 0.002) and endothelial (r = –0.49; p = 0.02) frequency ranges. The level of serum VEGF increases with decreasing IPV-1 in the endothelial range (Fig. 2, right).

Fig.2

Indices of vasoconstriction (IVC) in the endothelial range vs the MAU level (left), and indices of post-cold vasodilatation-1 (IPV-1) in the endothelial range vs the VEGF level (right).

4 Discussion

The cold test causes a powerful activation of the sympathetic nervous system, resulting in vasoconstriction. A massive stimulation of thermoreceptors during exposure to cold leads to activation of the sympathetic tone and a moderate increase of catecholamines in the blood plasma, but does not increase the frequency of the heartbeat. These processes may cause vasoconstriction (in arteries, arterioles and arteriovenous anastomoses), decrease in skin blood flow and possibly raise the arterial blood pressure [39, 40]. A significant part of the local cooling effect is due to vasoconstriction through the endothelial mechanism of regulation by inhibition of the NO synthesis. Furthermore, it is known that the initial period of vasoconstriction during local cooling is associated with the Rho–Rho kinase system, which evidently operates through mobilization of a2c receptors. Probably, the next period of vasoconstriction includes both the activation of adrenergic receptors and inhibition of NO-synthase. It has been found that blocking the NO system and adrenergic system with L-NAME and Bretylium, respectively, prevents vasoconstriction during the cooling test. The contralateral cold pressure test excludes a direct influence on the vessels of the hand and indirectly provide strong vasoconstriction, which was first described by Lewis in 1930. Cutaneous receptors stimulated by the ice-water immersion of one hand increase sympathetic nerve activity to the palm skin in the nonimmersed contralateral hand and reduce the blood flow, which is reflected as a decrease in skin surface temperature under a constant ambient environment [41, 42].

A vasoconstrictor response to cold in healthy subjects is accompanied by a decrease in the amplitude of skin temperature oscillations. After cold exposure, there occurs a postcold vasodilation, which is accompanied by an increase in the amplitude of skin temperature oscillations up to the initial values within different frequency ranges. Thus, such a reaction can be considered as an adequate response to the physiological pressor test due to preservation of the endothelial cell vasodilator function. An adequate response of the vascular tone to cold stress in the control group with normal levels of VEGF and MAU was characterized by the average values of the indices of vasoconstriction of 0.54±0.24 and the indices of vasodilation in the interval of 1.0±0.55.

However, in MetS patients, after the reduction in the amplitudes of the oscillations, the final recovery of these amplitudes in the endothelial and neurogenic frequency ranges was absent. In this group, the index of post-cold vasodilation had much lower values compared to the control group, and this indicates the lack of recovery of the amplitudes of oscillations due to violations of the vasodilatory mechanisms, which can be considered the hallmark of endothelial dysfunction [32]. Furthermore, the level of MAU was directly correlated with the value of the index of vasoconstriction, and the level of VEGF was negatively correlated with the value of the index of vasodilation-1 in the MetS group.

Patients with MetS exhibited significant differences from healthy subjects in all metabolic parameters. The results suggest that in this group of patients, hypertension, hyperuricemia, dyslipidemia, hyperglycemia and hyperinsulinemia are potentially endothelium-damaging agents. Accordingly, the potential mechanisms of MetS damage on vascular endothelial function are as follows [43]: 1) disturbance in carbohydrate metabolism (hyperinsulinemia/insulin resistance or hyperglycemia/diabetes), dyslipidemia, obesity and hypertension, which can all cause alterations in vascular contractility and thereby increase diastolic dysfunction through a variety of channels; 2) under normal conditions, endothelial cells can prevent thrombosis due to their anticoagulant properties; 3) when hyperglycemia, hyperlipemia, and hypertension occur, the release of various cytokines such as interleukin-1 (IL-1), IL-6 and platelet-derived growth factor (PDGF) are increased, which can promote the migration and proliferation of vascular smooth muscle cells, and also play an important role in the development of endothelial dysfunction process; 4) The sympathetic nervous system may directly influence the endothelium. Endothelial cells possess both α2-adrenoceptors and β-adrenoceptors [44]. Given that activation of endothelial adrenergic receptors releases endothelium-derived relaxant factors like NO and endothelium-derived contracting factors such as ET-1, the disturbances in the balanced release of these factors as a consequence of altered activity of the sympathetic nervous system may explain the role of this system in abnormalities of endothelial function in obesity-associated hypertension. The best evidence of this comes from studies showing that exaggerated sympathetic nervous system activation may impair endothelial function [45].

Our data confirm the existence of a relationship between the factors of endothelial dysfunction and metabolic abnormalities in ÌetS patients. The level of insulin was associated with the index of vasoconstriction in the endothelial and neurogenic ranges. Insulin has a very strong stimulating effect on the expression of the eNOS gene and the activity of this important enzyme. In the case of insulin resistance, the effect of insulin mediated through PI-3-kinase is significantly inhibited, and this reduces its impact on the activity of eNOS and on the production of NO. It has been found that under these conditions insulin loses its ability to maintain a stable state of smooth muscle cells and, conversely, begins to stimulate their proliferation and migration through the undamaged MAP-kinase pathway, i.e. it affects their capability to act in a pro-atherogenic manner [39]. Thus, the significant contribution of insulinemia to the development and maintenance of an endothelial dysfunction was confirmed for MetS.

Serum VEGF and microalbuminuria levels in the MetS group were significantly higher than those in the control group. Under hypoxic conditions, the endothelium starts expressing VEGF, which normally contributes to vasodilation due to the activation of prostacyclin and the stimulation of NO production, and launches angiogenesis. Although there are only few investigations of the interaction between the VEGF level and the vascular tone, they do show that VEGF causes variable dose dependent effects on arterioles with normal tone, but a potent vasodilatation in arterioles pre-contracted with endothelin-1, effects of VEGF dilatation are significantly counteracted by the anti-VEGF agent bevacizumab [9].

Serum VEGF and MAU levels were positively associated with TC, uric acid, SBP and DBP. In our work, a correlation between the levels of VEGF and DBP has been established, and this confirms the presence of endothelial dysfunction in patients with a short duration and low degree of hypertension. Angiotensin 2, in addition to hypoxia, also stimulates the production of VEGF. A number of studies have shown a significant increase in the VEGF in young patients with short arterial hypertension duration [46].

It has been found that the renin-angiotensin system (RAS) exists in different organs and tissues in MetS, and long-term hyperglycemia and hyperinsulinemia may increase PAC [47]. Several peptides involved in the RAS have been implicated in insulin resistance [48 –50] or hypertension [51, 52]. Hypercholesterolemia can also increase AT1R gene expression on vascular smooth muscle cells [53]. Low-density lipoprotein receptor-deficient mice fed a diet enriched in fat and cholesterol exhibited elevated plasma concentrations of angiotensinogen and angiotensin II [54]. Moreover, angiotensin may reduce the amount of glucose consumed by the organism and its insulin sensitivity, increase insulin resistance in skeletal muscles and adipose tissue, and inhibit signal transmission, decreasing thereby insulin effects.

An imbalance between vasoconstriction and vasodilation leads to disturbances in the maintenance of vascular tone and the function of vessels and their structure, which contributes to the development of hypertension and atherosclerosis [55].

In some works, it has been shown that the level of VEGF in obese patients increases significantly compared to patients with a normal BMI, which is considered as a response to hypoxia and angiogenesis activation [56]. We also found a significant increase in the VEGF in the group with a high BMI index. Moreover, we revealed a direct correlation between the level of VEGF and the index HOMA. Thus, an increased level of VEGF in MetS patients is a significant marker of endothelial dysfunction, and is responsible for the progression of hypertension and insulin resistance.

At present there are many research studies, in which the authors discuss the possibility of recovering endothelial function under treatment. In [57], it is shown that statins and fibrates have a positive impact on endothelial function. Statins are able to modulate a series of processes leading to reduction of the accumulation of esterified cholesterol in macrophages, increase of endothelial NO-synthetase, reduction of inflammation, increase of the strength of atherosclerotic plaques, and recovery of platelet activity and coagulation. Based on the results described in [58], it can be concluded that treatment with the AT1 (angiotensin type 1) receptor antagonist losartan improves the structural abnormalities and normalized endothelial function of small arteries from patients with mild to moderate essential hypertension. Blockade of AT1 receptors with attenuated angiotensin II–induced oxidative stress can decrease the degradation of nitric oxide and results in improved endothelial function. Reflex elevation of plasma angiotensin II elicited by AT1 antagonists and stimulation of unblocked AT2 receptors can favorably affect the endothelium, as in the kidney, where AT2 receptor stimulation elicits the generation of nitric oxide. This is similar to the improvement of endothelial function in resistance arteries from hypertensive patients under ACEI (Angiotensin I–converting enzyme inhibitors) therapy. ACEI treatment improves endothelial dysfunction of epicardial coronary arteries in patients with coronary artery disease [59]. These medications are commonly used to treat MetS. However we included in our study subjects with a newly diagnosed metabolic syndrome, who did not take earlier antihypertensive drugs, statins and other medical products exerting their effect on endothelium.

The violations of vasodilation diagnosed in MetS patients during the functional cold test confirmed the existence of a relationship between the abnormal vascular reactivity and the markers of endothelial dysfunction. An increased level of VEGF, which normally provides vasodilation in response to a spasm under pathology conditions, does not lead to an expansion of vessels, possibly due to the depletion of NO in endothelial cells. However, the level of VEGF remains high, and this likely reflects the launch of another mechanism involved in eliminating hypoxia, namely angiogenesis. Therefore, in the case of MetS, an increase in the levels of endothelial dysfunction biomarkers is accompanied by violation of the vasoconstriction mechanisms and reduction of the vasodilation activity. Thus, our data support the fact that in the case of MetS there is an imbalance between the factors that ensure optimal implementation of all the endothelium-dependent processes. This imbalance can be identified by the WAST technique.

4.1 Study limitations

Special emphasis should be placed on the limitations inevitably occurring in our investigations. First, the sample size was relatively small. Secondly, the study was mainly done on male population; no gender differences were taken into account during the cold pressor test. However, as it is shown in [26], the blood flow dynamics and cardiovascular reactions such as endothelium-dependent vasodilation are related to gender differences.

5 Conclusion

The results obtained in our investigation through use of biochemical markers and innovative instrumental techniques provided evidence in support of the available data indicating the presence of endothelial dysfunction in patients with MeTS. The WAST techniques were correlated with the biomarkers of endothelial dysfunction, and the possibility of their use for the diagnosis of functional disorders of endothelium was confirmed. The correlation of the values of vasoconstriction and vasodilation indices with the main factors of the metabolic syndrome demonstrated the close relationship between the biological and functional aspects of endothelial dysfunction.

Footnotes

Acknowledgments

The work was supported by the Russian Science Foundation under project 14-15-00809.

The authors have no conflicts of interest to declare.

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