The diagnostic value of some biomarkers in the assessment of endothelial dysfunction in chronic liver diseases: literature review

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Introduction

A current priority in modern hepatology is the study of pathogenetic mechanisms underlying chronic liver diseases (CLD). In this context, significant emphasis is placed on endothelial dysfunction (ED), which is now regarded as one of the early manifestations of many diseases and as a universal pathogenetic mechanism. Endothelial cells lining blood vessels are located between circulating blood and tissues, making them highly vulnerable to the effects of free radicals, metabolic byproducts, pathogenic factors, and medications that cause endothelial damage and dysfunction, impairing the production of biological substances [1]. Today, there are numerous laboratory and instrumental methods for assessing the functional activity of the endothelium. Determining in fluids and tissues factors synthesized by the endothelium is of particular interest [1; 2].

The objective of the study is to characterize the physiological role of the endothelium and to assess the diagnostic significance of certain biomarkers associated with ED in CLD.

Physiological Role of the Endothelium

The endothelium is a system of cells with a surface area of approximately 350 m² that participates in vital functions. It secretes a vast number of biologically active substances (BAS), regulates vascular tone, vasculogenesis and angiogenesis, perfusion and microcirculation, as well as hemostasis and inflammatory responses [3]. It is important to note that there are significant phenotypic differences between endothelial cells (ECs) in different parts of the vascular system. Thus, cells from different locations in the same individual do not only express different surface antigens and receptors but may also respond differently to the same stimulus.

Five specialized forms of endothelial cells are distinguished: somatic, fenestrated, sinusoidal, lattice-like, and high endothelium of postcapillary venules [1]. The formation of the endothelial phenotype, in addition to genetic factors, is determined by the influence of hemodynamic factors, microenvironment, interaction with other cells, as well as the size, structure, biochemical organization and functions of the organ [4; 5]. ECs respond to a wide range of mediators, including cytokines, growth factors, adhesion molecules, vasoactive substances and chemokines, which affect numerous different cells. The endothelium serves as a barrier preventing free penetration of molecules and cells from the blood into the underlying interstitium and tissues. Normally functioning endothelium helps to maintain a balance between the processes of vasodilation and vasoconstriction, antithrombosis and prothrombosis, growth inhibitors and stimulators, pro- and anti-inflammatory factors, antioxidants and prooxidants (Fig. 1).

 

Fig. 1. Factors synthesized in the endothelium [1; 3; 6]

 

In summary, the functions of the endothelium can be described as following: synthesis of vasoactive factors, anticoagulant properties, enzymatic activity, participation in immune responses, and angiogenesis [7]. The assessment of endothelial functional activity, both under normal conditions and during pathological processes, can be performed by measuring the levels of the aforementioned BAS in the blood.

Features of Liver Hemocapillary Endothelium

The liver parenchyma contains an enormous number of hemocapillaries, resulting in slow blood flow within hepatic lobules that facilitates efficient exchange of substances between blood and liver cells. The intensity of hematotissue exchange and hepatocyte morphology largely depend on the condition of sinusoids¹. Liver sinusoidal endothelial cells (LSECs) are strategically positioned at the blood-liver parenchyma interface. These highly specialized cells are characterized by the presence of intercellular pores called fenestrae (0.1 μm in diameter), forming sieve plates that serve as a biological graded barrier between sinusoidal blood and the plasma filling the space of Disse (Fig. 2). Under physiological conditions, LSECs maintain liver homeostasis, exhibiting anti-inflammatory and antifibrogenic properties by preventing activation of Kupffer cells and hepatic stellate cells, while regulating intrahepatic vascular resistance and portal pressure [8; 9]. The hepatic sinusoid has a dual blood supply, receiving blood flow from the portal vein (70 %) and hepatic artery (30 %) [10; 11]. In the liver, as in other vascular beds, endothelial cells can produce vasodilators in response to increased shear stress to reduce blood pressure [10].

 

Fig. 2. Liver sinusoidal endothelium [13]

 

ECs are the main source of nitric oxide (NO) in the normal liver, which is generated by eNOS activated by shear stress, induce downregulation of vasoconstrictor molecules including ET-1, and participate in the regulation of vascular pressure in the liver [14]. Other molecules released by ECs that regulate blood flow include the vasodilator CO and cyclooxygenase pathway metabolites (thromboxane A2, prostacyclin), which paracrinally affect hepatocytes in the space of Disse, causing a decrease in blood pressure [15]. In 2000, Limmer et al. discovered that liver sinusoidal ECs have an immunomodulatory function [16].

The structural and functional characteristics of liver hemocapillary endothelium may be influenced by gender and age factors. Based on a multilevel computational model of liver metabolism, it has been established that the livers of women and men are metabolically distinct, which may affect disease progression and outcomes [17]. With age, liver hemocapillaries show decreased blood flow intensity and metabolic processes, along with increased secretion of inflammation-inducing factors. Kupffer cells are attached to the endothelium and become activated during infections and injuries. Hepatic stellate cells are located in the subendothelial space of Disse, participate in blood flow regulation, and therefore may influence the development of portal hypertension, contracting in response to ET-1².

Characteristics and Types of Endothelial Dysfunction

The main factors influencing endothelial functional activity include blood gas composition, hemodynamics (pressure and shear rate), hormones, mediators, cytokines, lipoproteins, and endotoxins. Two stages of EC activation are distinguished: type 1 activation and type 11 activation [1]:

• Type 1 activation (rapid) – EC contraction, increased interendothelial gaps, P-selectin expression, release of von Willebrand factor and tissue plasminogen activator into plasma;
• Type 11 activation (slow) – ECs express E-selectin on their surface, synthesize cytokines, monocyte chemoattractant protein 1 (MCP 1), and platelet-activating factor (PAF).

It is considered that under normal conditions, the elimination of EC activation stimuli (for example, removal of bacterial and viral endotoxins, reduction of cholesterol, elimination of ethanol metabolites, etc.) contributes to gradual restoration of the endothelium. However, during prolonged EC stimulation, various changes occur (morphological, biochemical, etc.), leading to structural and functional rearrangements, and further to irreversible EC damage [18].

Thus, chronic endothelial activation may lead to the formation of a "vicious circle" and the development of ED, which is described from the pathophysiological perspective as a "triad":

1. Shift in the balance of antagonist regulators.
2. Disruption of reciprocal interactions in feedback systems.
3. Formation of metabolic and regulatory "vicious circles" that alter the functional state of endothelial cells, leading to impaired tissue and organ function.

From modern perspectives, ED is a multifaceted process whose key mechanisms are: disruption of vascular wall integrity, imbalance between anticoagulants and procoagulants with formation of a prothrombotic phenotype, activation of cytokine and adhesion molecule production, vascular proliferation, leading to vascular wall remodeling [19–22].

However, the manifestations and severity of ED, characterized by impaired production of endothelial factors in various diseases, differ, which is associated with endothelial heterogeneity and depends on the structure, biochemical organization and function of the organ. Therefore, despite the "universality" of this pathogenetic mechanism, there are individual manifestations determined by nosology, which will result in varying degrees of impairment in the production and changes in concentrations of BAS. Based on the main functions of the endothelium, four main types of ED are distinguished [1]: vasomotor, hemostatic, angiogenic, and adhesive.

Chronic liver damage leads to profound dedifferentiation of ECs, characterized by loss of fenestration and development of basement membrane, which impairs normal lipoprotein metabolism and oxygen exchange, resulting in apoptosis. Decreased NO biosynthesis coupled with increased NO uptake leads to activation of hepatic stellate cells and extracellular matrix deposition, while overproduction of vasoconstrictors and proinflammatory cytokines exacerbates hepatic sinusoidal constriction. These pathological changes cause microvascular dysfunction, fibrosis, and ultimately lead to portal hypertension and cirrhosis [23–27]. Progressive liver fibrosis in hepatitis C virus infection also contributes to atherosclerosis development by inducing ED independent of general cardiovascular risk factors [28]. Furthermore, progression of endothelium-dependent endothelial dysfunction correlates with liver fibrosis progression in chronic hepatitis C [29].

Dysfunction of sinusoidal ECs is also involved in the progression of non-alcoholic fatty liver disease (NAFLD) through numerous mechanisms, including regulation of the inflammatory process, activation of hepatic stellate cells, increased vascular resistance, and impaired microcirculation. The liver releases inflammatory molecules that are considered proatherogenic and may contribute to vascular endothelial dysfunction leading to atherosclerosis and cardiovascular diseases [30]. Capillarization of sinusoidal ECs occurs at the earliest stage of NAFLD. Phenotypic changes in ECs promote the development of hepatic steatosis by preventing the release of very low-density lipoproteins from hepatocytes into the hepatic sinusoid, leading to increased accumulation of total cholesterol and triglycerides in the liver, as well as the production of lipids de novo in the liver, which contribute to hyperlipidemia and early pathological changes [31; 32].

The Value of Certain Biomarkers in Assessing ED in CLD

In CLD, a promising method for assessing the presence and severity of ED is the laboratory evaluation of BAS levels in blood produced by the endothelium [33]. In scientific research and partly in clinical practice, such ED biomarkers are used as stable nitric oxide metabolites (NO), endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), von Willebrand factor (vWF), monocyte chemoattractant protein-1 (MCP-1), measurement of pro- and anti-inflammatory interleukin levels and tumor necrosis factor alpha (TNF-α), and circulating EC count. New biomarkers (syndecans, endoglin, endocan) may also find clinical application in ED assessment in various diseases in the future.

NO represents a potent signaling molecule with vasodilatory and anti-inflammatory properties. This molecule is produced by the action of NO synthases and plays a key role in maintaining vascular homeostasis. In ED, there is a decreased capacity for NO production or reduced vascular wall sensitivity to it, consequently leading to increased vascular tone.

As a result of this process, the balance between vasodilation and vasoconstriction is disrupted, which can lead to various pathological conditions. The walls of blood vessels become stiffer and less elastic, increasing the risk of thrombus formation and inflammatory reactions [34]. The instability of this molecule limits the use of this biomarker in clinical practice, as it requires additional stabilization methods, complicating the technology and increasing the cost of the study. More widely used are techniques for assessing the levels of stable NO metabolites. Determination of nitric oxide in blood has been proposed for differentiating hepatitis and liver cirrhosis³. Experimental models of steatosis have demonstrated that NO participates in modulating hepatic microcirculatory perfusion and oxygenation [35]. Clinical studies have noted a significant increase in NO metabolite levels in patients with non-alcoholic steatohepatitis [36].

ET-1 is a potent vasoconstrictor produced by ECs that acts on smooth muscle cells by activating G-protein-coupled receptors and participates in regulating various metabolic processes. Endothelin has three peptide isoforms: ET-1, ET-2, and ET-3, which contain domains for expression and binding to ET(A) and ET(B) receptors. ECs produce more ET in response to oxidative stress, hypoxia, mechanical vascular damage, presence of oxidized low-density lipoproteins, and proinflammatory cytokines, leading to vasoconstriction and activation of fibrotic processes. Following the discovery of ET-1 as a potent vasoconstrictor, significant interest has emerged in its role in vascular function in liver diseases [37; 38]. It should be noted that measuring ET in blood involves certain preanalytical and analytical peculiarities that currently limit its widespread application.

vWF is synthesized by ECs and serves as a sensitive marker of endothelial damage. According to recent studies, dynamic assessment of its plasma levels helps predict mortality in hospitalized patients with viral infections [39]. In CLD, increased vWF activity is observed, being more pronounced in fibrosis [40]. Measurement of vWF functional activity in plasma has been proposed for differentiating hepatitis and liver cirrhosis [41]. In chronic hepatitis C, NO deficiency is accompanied by elevated ET-1 concentrations and increased vWF activity in blood. High vWF levels are associated with sinusoidal endothelial destruction and platelet-vascular hemostasis dysfunction [42]. Other earlier studies demonstrated that elevated NO and vWF levels - correlating with disease severity - may indicate endothelial dysfunction in liver cirrhosis [43].

VEGF is a glycoprotein with five isoforms differing in biological activity. The VEGFА isoform is more active in terms of blood vessel growth. This factor is a crucial regulator of angiogenesis, vascular permeability, and by interacting with VEGFR1 and VEGFR2 receptors expressed in many tissues, regulates cell growth, survival and metabolism [44, 45]. VEGF is necessary for stable physiological angiogenesis, but in a number of diseases becomes a key factor of pathological angiogenesis and ED [46]. The course of steatosis and viral liver fibrosis is characterized by ED development associated with VEGF overproduction, more pronounced in fibrosis and correlating with its progression rate [47; 48]. VEGF also plays a significant role in the pathogenesis of liver steatosis and dyslipidemia in patients with metabolic syndrome [49]. Moreover, its production significantly increases in severe obesity [50; 51]. VEGF, along with other biometric and laboratory parameters, is included in the calculation formula for diagnosing liver steatosis, whose sensitivity and specificity are 95.2 % and 97.0 % respectively [52].

Another method for determining endothelial status involves counting circulating ECs and evaluating their morphology. It has been shown that the degree of endothelial damage correlates with an increase in the number of circulating ECs in peripheral circulation, which reflect reparative potential [1]. In viral liver pathology and non-alcoholic fatty liver disease, an increase in the number of these markers has been recorded, indicating endothelial damage [8; 51]. To assess the number of circulating ECs, the J. Hladovec method modified by N.N. Petrishchev is used, as well as the flow cytometry method that allows evaluating the number of EC progenitor cells [1]. Thus, quantitative and qualitative assessment of mature circulating ECs and endothelial progenitor cells can be considered as a specific marker of ED, and also characterize the activity of reparative processes in the endothelium.

In recent years, a number of new biomarkers have emerged to assess endothelial function. These include factors characterizing the barrier function of the endothelium, its adhesive capacity and permeability. For example, cell adhesion molecules (E- and P-selectins), intercellular and vascular adhesion molecules ICAM-1 and VCAM-1, which are expressed under the influence of proinflammatory cytokines and modified lipoproteins, characterizing not only ED but also the activity of the inflammatory response [53]. Among the new markers of ED are also endocan, endoglin and the syndecan family, characterizing inflammatory processes associated with the endothelium [45; 54].

Non-invasive assessment of endothelial function also includes various instrumental methods that evaluate vascular elasticity, tone, and microcirculatory disturbances: photoplethysmography, laser Doppler flowmetry, and recording of skin temperature fluctuations during local heating with calculation of the thermal vasodilation index [54–56]. Specifically, in patients with hepatic steatosis and menopausal metabolic syndrome, decreased thermal vasodilation index values were observed, progressively worsening with increasing obesity severity [57]. According to several researchers, combining laboratory and instrumental methods provides more comprehensive information when assessing endothelial functional status [54; 58].

Conclusions

The analysis presented in this review, including results from our own studies, demonstrates the diagnostic value and practical utility of various biomarkers for assessing the presence and severity of ED in CLD. However, it should be noted that not all factors are strictly specific to the endothelium and may be elevated in comorbid pathology. There are also certain difficulties in performing tests and interpreting results for some biomarkers, in addition to the high cost of some reagents. Therefore, for a more effective evaluation of ED in CLD and correct selection of endothelial-protective therapy, an integrated approach using laboratory and instrumental methods or the use of several biomarkers reflecting different endothelial functions is required.

 

¹ Bulatova I.A. [Functional state of endothelium and its diagnostic significance in assessing severity of chronic diffuse liver diseases]. Abstract of Dissertation for the PhD (Medicine) Degree. Ural State Medical Academy. Yekaterinburg; 2009. Available at: http://elib.usma.ru/bitstream/usma/751/1/USMU_Thesis_2009_006.pdf

² Bulatova I.A. [Functional state of endothelium and its diagnostic significance in assessing severity of chronic diffuse liver diseases]. Abstract of Dissertation for the PhD (Medicine) Degree. Ural State Medical Academy. Yekaterinburg; 2009. Available at: http://elib.usma.ru/bitstream/usma/751/1/USMU_Thesis_2009_006.pdf

³ Shchekotov V.V., Shchekotova A.P., Bulatova I.A., Mugatarov I.N. [Method for differential diagnosis of chronic viral hepatitis and liver cirrhosis]. Patent RU 2383021 C1, 27.02.2010. Application No. 2008147941/15 dated 04.12.2008. (In Russian)

About the authors

I. A. Bulatova

Ye.A. Vagner Perm State Medical University

Author for correspondence.
Email: bula.1977@mail.ru
ORCID iD: 0000-0002-7802-4796

DSc (Medicine), Head of the Department of Normal Physiology, Professor of the Department of Faculty Therapy № 1

Russian Federation, Perm

T. P. Shevlyukova

Tyumen State Medical University

Email: bula.1977@mail.ru
ORCID iD: 0000-0002-7019-6630

DSc (Medicine), Professor of the Department of Obstetrics and Gynecology of the Institute of Maternity and Childhood

Russian Federation, Tyumen

A. P. Shchekotova

Ye.A. Vagner Perm State Medical University

Email: bula.1977@mail.ru
ORCID iD: 0000-0003-0298-2928

DSc (Medicine), Professor of the Department of Faculty Therapy № 2, Occupational Pathology and Clinical Laboratory Diagnostics

Russian Federation, Perm

E. N. Smirnova

Ye.A. Vagner Perm State Medical University

Email: bula.1977@mail.ru
ORCID iD: 0000-0003-2727-5226

DSc (Medicine), Head of the Department of Endocrinology and Clinical Pharmacology

Russian Federation, Perm

S. V. Paducheva

Ye.A. Vagner Perm State Medical University

Email: bula.1977@mail.ru
ORCID iD: 0000-0001-8255-088X

PhD (Medicine), Lecturer of Medical and Pharmaceutical College

Russian Federation, Perm

I. V. Shchekotova

Contract research organization LLC "Synergy"

Email: bula.1977@mail.ru

Head of the Customer Service Department

Russian Federation, Moscow

N. S. Bessonova

Tyumen State Medical University

Email: bula.1977@mail.ru
ORCID iD: 0009-0008-3821-7252

PhD (Biology), Associate Professor of the Department of Chemistry and Pharmacognosy

Russian Federation, Tyumen

References

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