Methods for diagnosing endothelial dysfunction

Endothelial dysfunction as a crucial factor in the pathogenesis of cardiovascular diseases requires precise and effective diagnostic methods. The review highlights the currently used methods which can be divided into morphological, instrumental, and laboratory ones. Special attention is paid to the so called jar test, which was introduced by Professor V.A. Valdman in 1936. The jar test may serve as a prototype of modern methods for endothelial function assessment. These diagnostic methods can help to identify functional endothelial disorders at the earliest stage. It will significantly expand the possibilities of primary prevention of cardiovascular and a number of other diseases through non-pharmacological and pharmacological correction of endothelial dysfunction. Conflict of interest. The authors declare the absence of obvious or potential conflict of interest related to the publication of article.


INTRODUCTION
The endothelium is a thin layer of cells that line the interior surface of blood vessels, lymphatic vessels, and the endocardium, which implements coordination and an optimal course of diverse metabolic and physiological processes [1]. As the most important regulatory and endocrine organ, the endothelium performs such crucial functions as control over leukocyte adhesion, vascular tone, and angiogenesis, regulation of platelet adhesion and aggregation, and involvement in fibrinolysis and inflammatory processes [2].
Endothelial dysfunction is one of the most important links in the pathogenesis of cardiovascular diseases. The relevance of the problem implies introduction of efficient and accurate diagnostic methods that can help identify functional disruptions of the endothelium at the earliest stage. It will significantly expand the possibilities of primary prevention of cardiovascular and a number of other diseases through non-pharmacological and pharmacological correction of endothelial dysfunction.
The currently used methods for the endothelial function can be conditionally divided into morphological, instrumental, and laboratory ones.

MORPHOLOGICAL METHODS
The so called jar test, which was proposed back in the 1930s, can serve as a prototype of morphological methods for endothelial function assessment. Professor V.A. Valdman, the founder and the first head of the Department of Intermediate Level Therapy of the Leningrad Pediatric Medical Institute, was one of the first to call the vascular endothelium "the arena where the first collision of a macroorganism with microbes occurs". Studying vascular pathology, in 1936, he proposed a test as a method for detecting hyperergic swelling of the vascular endothelium (endotheliosis) [3]. In fact, the jar test can be rightfully considered as one of the first attempts to assess the functional state of the vascular endothelium.
In 1978, J. Hladovec introduced a method for assessing endothelial dysfunction by examining the level of circulating desquamated endothelial cells (DEC), which are cells that separate from the vessel wall during its damage [4]. Currently, a computer-assisted analysis of a DEC image is carried out (the cytometry method). On average, the amount of DEC in adults normally varies from 2 to 4 cells / 100 µl of plasma [5]. In patients with cardiovascular diseases, it was proposed to distinguish the degree of endothelial dysfunction by the DEC concentration. A moderate degree of endothelial dysfunction is defined when the number of DEC is from 6 to 10, an average degree is from 11 to 25, and a pronounced degree -26 or more [6].
Recently, increased attention has been paid to apoptotic endothelial microparticles (EMPs) as new markers of endothelial dysfunction. EMPs are membrane vesicles released into the extracellular space during activation or apoptosis of endothelial cells [7]. EMPs are known to express markers of cell damage, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and von Willebrand factor (vWF) [8]. The EMP concentration is determined by flow cytometry, and their morphology and size are determined by cryotransmission electron microscopy using receptor-specific labeling [9].

INSTRUMENTAL METHODS
One of the first instrumental methods to estimate the vasoregulatory function of the endothelium was coronary angiography with the administration of Shabrov A.V., Galenko A.S., Uspensky Yu.P., Loseva K.A.
Methods for diagnosing endothelial dysfunction acetylcholine for evaluating endothelium-dependent vasodilation (EDVD) and sodium nitroprusside for evaluating endothelium-independent vasodilation (EIVD) (S.W. Werns et al., 1989) [10]. Usually, acetylcholine is used for intracoronary administration during coronary angiography; interacting with the intact endothelium, it has a stimulating effect on the production of nitric oxide (NO) and other endothelium-derived relaxing factors. In endothelial dysfunction, or damaged endothelium, acetylcholine causes either weak vasodilation or even vasoconstriction [11]. To assess EIVD, exogenous sources of NO (nitroglycerin, sodium nitroprusside), which directly affect vascular smooth muscles, are administered via the intracoronary route. Changes in the diameter of the arteries are recorded using digital quantitative image analysis systems and intravascular ultrasound catheters [12]. The method is the most accurate for assessing the vasoregulatory function of the endothelium due to direct assessment of vascular reactivity in the coronary arteries. However, it has limitations due to the invasiveness of the procedure and the high cost.
D.S. Celermajer et al. (1992) developed a non-invasive method for diagnosing the functional state of the endothelium by evaluating postocclusive changes in the diameter of the brachial artery using highresolution ultrasound [13]. This method for evaluating endothelial function is called flow-mediated dilation (FMD). During the test, endothelium-mediated NO release occurs, which leads to vasodilation. The degree of vasodilation can be quantified as an indicator of vasomotor function [14]. The method consists in an increase in shear stress as a result of elevated blood flow through the peripheral artery. This is achieved by creating reactive hyperemia, which implies a five-minute occlusion of the brachial artery by inflating the cuff to a pressure 50 mmHg higher than systolic blood pressure. Then, after five minutes, the cuff is decompressed, which leads to increased blood flow and increased shear stress. Using a high-resolution ultrasound sensor, blood flow velocity parameters are measured within 15-20 seconds, and the artery diameter is estimated 45-60 seconds after the decompression, when its maximum increase is noted [13]. The EDVD index of the brachial artery is calculated by the formula: where D 1 is the initial diameter of the brachial artery, and D 2 is the diameter of the brachial artery after de-compression [15]. The advantage of the method is its non-invasiveness. It allows to repeat measurements to evaluate the effectiveness of various interventions that may affect the state of the vascular endothelium [16].
T. Anderson et al. (1995) demonstrated a close correlation between the EDVD parameters obtained during coronary angiography with acetylcholine and parameters obtained during ultrasound examination of the brachial artery. It allowed to use the brachial artery as a universal model for further evaluation of the effects of various factors on endothelial function [17].
T. Gori et al. (2008) described a new non-invasive method -low flow-mediated constriction (L-FMC), which allows to assess arterial tone at rest. L-FMC consists in narrowing the brachial artery due to a decrease in blood flow after arterial occlusion using a distal cuff. L-FMC is a simple and fast method that complements FMD and extends diagnostic capabilities [18].
The simplest and fastest method for instrumental diagnosis of endothelial dysfunction is analysis of the volume pulse wave shape, which is recorded using a photoplethysmographic sensor located on the nail phalanx of the patient's finger, with subsequent processing of the received signal on the computer. During the study, the contour of a volume pulse wave is recorded by merging two waves: systolic (direct) and reflected. The reflection index (RI) is calculated as the ratio of direct and reflected wave amplitudes to the reflection time (T), by which the reflected wave lags behind the systolic one. The stiffness index (SI) is calculated as the ratio of the patient's height L (in meters) to the reflection time (in seconds). In various pathologies of the cardiovascular system, a decrease in vascular elasticity is observed, which is indicated by an increase in the RI and SI [19].

LABORATORY METHODS
Laboratory methods for evaluating endothelial function consist in determining the concentration of certain factors synthesized by the endothelium and performing various functions.
Mainly, the levels of NO and its metabolites are primarily used to assess the endothelial vasomotor function [20]. It is known that NO is synthesized as a result of oxidation of L-arginine by the oxygen atom with the participation of NO-synthase: 2L-arginine + 3NADPH + 4O 2 + 3H+ → 2L-citrulline + 2NO + 3NADPH + 4H 2 O According to their structure, NO-synthase (NOS) isoforms are divided into endothelial (eNOS), neuro-Бюллетень сибирской медицины. 2021; 20 (2): 202-209 nal (nNOS), and inducible (iNOS). According to the mechanism of action, they are divided into constitutional (cNOS) and inducible (iNOS) [21,22]. cNOS are involved in NO synthesis in hypoxic conditions with vasoconstriction of blood vessels, iNOS -after their induction by bacterial endotoxins, some inflammatory mediators, and reactive oxygen species (ROS) [21]. NO-synthases can exert their effect only in the presence of cofactors, such as flavins, NADPH oxidase (NOX), and tetrahydrobiopterin [23].
Since NO has a half-life of only 0.1 seconds [24], the levels of end products of NO oxidation -nitrites and nitrates (NO x ) -are usually determined. The study material can be plasma, blood serum, or culture fluids [25]. The most common method for determining the NO x levels is the spectrophotometric method with Griess reagent.
At the first stage of this study, the nitrite ion is determined by the reaction of nitrites with Griess reagent, which contains an aqueous solution of 0.05% N-(1-naphthyl)ethylenediamine and a 1% solution of sulfonamide in acetic acid [26]. The reaction produces a stained diazo compound with an absorption maximum at the wavelength of 548 nm. Since Griess reagent detects only nitrites, reactions of reducing nitrates to nitrites are performed using either nitrate reductase or Vanadium (III) [27,28].
In various cardiovascular diseases, a decrease in NO x concentration is observed, which may be caused by a decrease in the expression and activity of eNOS, a decrease in the level of L-arginine and NO-synthase cofactors, or an increase in endogenous NO inhibitors. In some patients, there is an increase in the level of NO x with functional signs of reduced EDVD, which indicates severe endothelial dysfunction. An increase in the concentration of the end products of NO oxidation may be associated with activation of iNOS by inflammatory mediators (tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β), lipopolysaccharides (LPS), and ROS. With excess NO, toxic peroxynitrite is formed, which has a cytostatic and cytotoxic effects [25].
To assess endothelial dysfunction, the concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, is also determined. ADMA is an amino acid synthesized from arginine by protein arginine methyltransferase (PRMT). There are two isoforms of PRMT, one of which, PRMT-1, activates ADMA formation, and the second one, PRMT-2, activates symmetric dimethylarginine (SDMA) formation. It is ADMA that has an inhibi-tory effect on three NOS isoforms [29]. The ADMA concentration is determined using high-performance liquid chromatography with highly sensitive laser-induced fluorescence detection [30].
To assess vasoconstrictor functions of the endothelium, the level of endothelin-1 (ET-1) is determined. High concentrations of ET-1 contribute to the development of endothelial dysfunction. ET-1 increases production of endothelial superoxide, which leads to EDVD disruption and triggering of proinflammatory processes [31]. High concentrations of ET-1 in the blood serum are found in patients with coronary artery disease, arterial hypertension, kidney disease, as well as obstetric and gynecologic pathology [32].
Evaluation of endothelial function also involves determining the concentration and functional activity of von Willebrand factor (vWF), which mediates platelet aggregation and adhesion to the vascular endothelium. vWF is a multimeric plasma glycoprotein that is excessively synthesized by endotheliocytes and megakaryocytes in the form of a propeptide (vWFpp), which then undergoes post-translational modifications. The vWFpp, together with vWF, is stored inside endothelial cells in Weibel-Palade bodies and in α-granules of megakaryocytes [33][34][35]. Inflammatory cytokines are able to stimulate production of vWF and vWFpp in high concentrations from Weibel-Palade bodies and α-granules (IL-8 and TNF-α) and inhibit the cleavage of vWF (IL-6). As a result, the hyperreactive factor accumulates in the blood and on the surface of endothelial cells, which leads to increased activation of platelet aggregation and adhesion to vascular endothelium [36]. Studies showed the importance of increasing the vWF concentration as a predictor of a risk of recurrent myocardial infarction [37].
There are several laboratory tests for assessing the level and activity of vWF. Quantitative determination of the vWF antigen is performed by the enzyme immunoassay (vWF:Ag) [34]. It is also proposed to evaluate the level of vWFpp in the blood plasma as a marker of endothelial dysfunction, since vWFpp is not used in platelet aggregation, in contrast to the vWF antigen (vWF:Ag) [38]. Analysis of the vWFpp / vWF:Ag ratio is used to assess the degree of vWF clearance. In normal human plasma, the vWFpp / vWF:Ag ratio is 1.0. In patients with type 1C von Willebrand disease, a decrease in the vWF concentration is observed due to its rapid elimination, which leads to an increase in this ratio [33,39].
Activity evaluation (ristocetin-cofactor (RCoF) activity) is based on the ability of vWF to activate platelet aggregation and adhesion through interaction of the factor A1 domain with the platelet membrane receptor -glycoprotein Ib (GPIb). The process is imitated by adding ristocetin to the suspension of washed red blood cells in the presence of the patient's plasma, which promotes binding of vWF to GPIb [40]. The aggregation rate is measured by an aggregator. Normal values of the method range from 50 to 200 IU / dl [41]. The test does not exclude false results due to possible defects in the ability of vWF to bind ristocetin [42]. A new analysis was developed that does not involve the use of ristocetin. It is based on the use of recombinant GPIb with enhanced function, which spontaneously binds vWF in vitro [43].
vWF is able to form bonds with collagen through the A3 domain when the endothelial lining is damaged. This fact is the basis for another diagnostic test -quantitative determination of the collagen-binding activity of vWF by enzyme immunoassay (vWF:CB) [44].
Through the domains D and D3, vWF binds to coagulation factor VIII (FVIII) and transports it through the bloodstream in an inactive form. If the endothelial lining is damaged, vWF delivers FVIII to the site of injury, where FVIII forms a complex with coagulation factor IX [34,40,41]. The vWF / FVIII binding test (vWF:FVIIIB), which is mainly used for the diagnosis of type 2N von Willebrand disease, allows to evaluate the affinity of vWF to FVIII [45].
To analyze the anticoagulant function of the endothelium, the level of thrombomodulin (TM), which is a transmembrane glycoprotein expressed on the surface of endothelial cells of blood vessels, is examined. At the site of endothelial damage, thrombin binds to TM, forming a thrombin -TM complex that promotes activation of protein C, which splits factors Va and VII-Ia, thereby preventing excessive fibrin clotting. The concentration of TM in the plasma of healthy people is relatively low. In case of damage to the endothelium, accompanied by proteolysis, the concentration of TM in the blood plasma increases by 1.5-2 times. The level of TM in the blood is measured by the enzyme immunoassay [46].
To assess fibrinolytic function, the level of tissue plasminogen activator (tPA) produced in endothelial cells is examined. The tPA provides an external pathway for plasminogen activation by forming a triple complex (fibrin + plasminogen + tPA), due to which the resulting plasmin provides proteolytic degradation of fibrin [47]. The tPA concentration is also determined by the enzyme immunoassay [32].
The endothelium is known to play an important role in adhesion and migration of leukocytes through secretion of adhesive molecules, such as ICAM-1, VCAM-1, P-selectin, E-selectin, and VE-cadherin. Changes in the concentration of these substances indicate endothelial damage and the severity of inflammation, and can be used to determine a prognosis of the disease [48].
The endothelium is directly involved in activation of vascular growth in hypoxia and tissue damage. One of the most important regulators of angiogenesis is vascular endothelial growth factor (VEGF), which is used as a marker of endothelial dysfunction. Determination of VEGF concentration is most important for the diagnosis of cancer, since the growth of blood vessels in a tumor contributes to its activation, rapid growth, and metastasis. VEGF, which interacts with vascular endothelial receptors, is the most powerful mediator of this process [49].

CONCLUSION
Currently, a clinical researcher has a wide range of methods for endothelial function assessment: laboratory, instrumental and morphological techniques. Further research of the endothelial function in normal and pathological conditions could clarify components of the pathogenesis of cardiovascular diseases that are still unclear and suggest measures of primary and secondary prevention. Parameters that characterize the functions of the endothelium, of course, are only surrogate intermediate points for evaluating the effectiveness of certain effects. However, on the other hand, the assessment of endothelial function can have a great prognostic value, since endothelium dysfunction is one of the earliest preclinical markers of vascular damage. And, conversely, restoration of impaired parameters is probably one of the first markers of the effectiveness of a preventive or therapeutic intervention. Therefore, the study of the properties and functions of the endothelium, as well as development and optimization of diagnostic methods for assessing its condition are necessary and relevant.