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ENDOCARDIAL ENDOTHELIUM AS A BLOOD-HEART BARRIER/ENDOKARDNIENDOTEL KAO KRVNO-SRCANA BARIJERA.

Abbreviations
EE       --endocardial endothelium
PECAM-1  --platelet-endothelial adhesion molecule-1
TRP      --transient receptor potential
ATPase   --adenosine triphosphatase


Introduction

The cardiovascular system is lined with endothelium, a continuous single-layer flaked epithelium, forming a cobblestone-like layer on the surface of the tunica intima of the blood vessels and the endocardial layer of the heart. It has a total surface area of several hundred square meters and the endothelium of a person weighing 70 kg covers about 700 [m.sup.2] [1]. The vascular endothelium has long been thought of as just a layer lining the blood vessels like a cellophane wrapper, without a significant functional role. It is now recognized as a massive, regionally specific multi-functional organ. Out of all the layers of the blood vessel wall, endothelium is the most exposed to mechanical forces and the pressure that blood exerts on it.

Given the strategic anatomical position of endothelium between the circulating blood components on one side and a vascular component of smooth muscle and cardiac muscle on the other, it represents a physiologically significant organ whose dysfunction is seen as a critical factor for the development of various diseases [2]. Before the initial research on the effects of endocardial endothelium (EE) on the contractility, rhythm and remodeling of the adjacent myocardium, information about any physiological roles of EE on the function of the heart were rare [3]. However, studies have shown that myocardial contractility depends on the presence of EE and the degree of its damage regardless of the technique employed to remove the endothelium: immersion in 1% Triton X-100, mechanical peeling, exposure to a high-frequency ultrasound, or flow rate of dry air. Fully removing or partially damaging EE cells directly affect contractile cardiac performance causing a lower contractility of cardiomyocytes [4, 5]. The inotropic effect of EE is achieved through the synthesis and release of endothelial mediators, the sensory ability to detect changes in blood plasma and the quality of bloodheart barrier to control transendothelial transport. Adding a mediator of endothelial origin will not restore or prevent the loss of heart contractility. The EE can affect the establishment of transcellular physicochemical gradient across the endocardium, and thereby affects the cardiac function [6].

The removal of EE is reflected in the acute disorder of subendocardial ionic environment. It took time and numerous studies to support the hypothesis of EE as the blood-heart barrier and to compare its importance to the one of the blood-brain barrier. In the last 20 years, following the initial studies of Paul Fransen (1995), the analogy of EE barrier with the concept of the blood-brain barrier has strengthened [7]. The blood-brain barrier is the best studied endothelial barrier. Highly excitable tissues such as neurons have a larger concentration of [Na.sup.+] ions in the interstitium, which enables a faster rise of action potential and less interstitial [K.sup.+], thereby increasing the membrane excitability. In the heart, the sub-endocardial network of terminal Purkinje fibers and the surrounding myocardial cells consist of highly excitable cells making the ion homeostasis the essence of the vitality of the heart function. Changes in the extracellular concentration of [Ca.sup.++], [K.sup.+], [Na.sup.+], [Mg.sup.++], [Cl.sup.-] i [HCO.sub.3.sub.-] ions have significant effects on the rhythm and the mechanical properties of the cardiac muscle.

The maintenance of the assumed trans-endocardial electrochemical potential differences provides a high gradient for certain ions while the selective barrier, basal lamina, prevents ionic leakage. The negatively charged glycocalyx also modulates endothelial permeability.

Morphological Characteristics of the Blood-Heart Barrier

The heart wall consists of three layers, epicardium, myocardium and endocardium. Each layer is distinctive in thickness, cellular composition and the specific role. From a histological point of view, endocardium consists of three layers: the endothelium, the subendothelial loose connective tissue and subendocardium.

The fibrous component of the subendocardium consists of a small amount of collagen and elastic fibers [8]. Electron microscope images clearly show the existence of typical telocytes within the subendothelial layer. Telocytes appear as the main interstitial cells in the loose connective tissue of the subendothelial endocardium. The subendocardium consists of telocytes, fibroblasts and nerve endings. About one third of endothelial cells are underlined by telocytes. Subendocardial telocytes have small oval-shaped cellular bodies with very long, thin cellular processes named telopodes. Telocytes are in close vicinity with nerve endings and they establish contacts with other interstitial cells in the subendocardial space. Telocytes create a tridimensional cardiac network at the interstitial level and integrate all constitutive layers of heart via their telopodes. Since endocardium is considered a blood-heart barrier, it is obvious that telocytes which constitute the main population in the subendothelial layer of endocardium may be of importance in creating this blood-heart barrier [9].

The EE forms a single layer of closely related cells with complex interrelations and extensive overlapping of adjacent edges. This structure allows its unique permeable properties. Tight junctions are located at the luminal side of intercellular gaps between endothelial cells. The EE cells exhibit characteristic morphological asymmetry.

Electrophysiological measurements of properties and electric currents, as well as fluorometric measurements, have demonstrated that EE cells form an interconnecting network through electrical synapses, such as gap junctions. Gap junction channels of EE cells are composed of different connexin types (Cx43, Cx40, Cx37) and are located at the borders of the intercellular areas and at the overlapping areas and the EE cells [10].

Gap junctions have intercellular pores that facilitate the passage of ions (mostly Ca ), secondary messenger molecules (inositol-1,4,5 trisphosphate) and small metabolites which pass without pausing between adjacent EE cells, ensuring that the entire EE acts as a single unit with electrical and diffusive continuity [11]. We examine the EE as a syncytium with electrochemical connections between neighboring EE cells. Gap junction connections between heart endothelial cells and cardiomyocytes are not identified. The absence of morphological connections between EE and cardiomyocytes does not exclude the electrotonic current propagation or the effects of EE syncytium on the excitability and conductivity to the adjacent cardiomyocytes.

Endocardial endothelium syncytium can be functionally represented as a large cell, with a very large membrane area, which is connected to smaller groups of Purkinje fibers and subendocardial nerve plexuses. Electrical phenomena arising from the high conductive power of cardiomyocyte syncytium may propagate to the Purkinje network, nerve plexus and EE.

There is no experimental evidence to support a conclusion that EE cells are "electrically silent" in the sense that they do not show regular action potentials. However, they depolarize and repolarize following the action potentials of adjacent cardiomyocytes [12]. Syncytial character of EE is essential to establish the barrier properties in the transcellular transport of ions. Moreover, it is possible that it enables an increased synthesis and the release of endothelial paracrine mediators.

Intercellular Junctions between EE Cells

Endothelial cells are connected by a complex set of junctional proteins that comprise tight junctions, adherens junctions and gap junctions. Gap junctions form transmembrane channels between contiguous cells. Tight junctions and adherens junctions form pericellular zipper like structures along the cell border through their transmembrane homophilic interactions.

The cell adhesion necessary to maintain the integrity of all tissues, including the endothelium, involves two groups of the adhesion proteins, in addition to intercellular junctions, hemidesmosomes, focal adhesions and extracellular matrix proteins. One group of adhesion proteins is responsible for the adhesion of cells with the extracellular matrix (integrins), while the other group participates in intercellular adhesion of endothelial cells. One very important adhesion protein is platelet-endothelial adhesion molecule-1 (PECAM-1) that is expressed on the surfaces of the interendothelial contacts. PECAM-1 is uniformly distributed along the juncture giving it stability and is one of the main components of the endothelial junctions. Identification of PECAM-1 enables visualization of intercellular junctions within endocardial cells. In the EE, PECAM-1 staining is typically confined to the border zone of the EE cells corresponding to the zone of cellular overlap and intercellular clefts.

Transendothelial transport (transcytosis) is mediated by diffusion through intracellular clefts by vesicular transport, or focal adhesion contracts (gap junctions). Thus, trans-EE-permeability is predominantly controlled through intracellular clefts, mediated through the extent and structurally complex paracellular space which is partially lined by an electrically charged glycocalyx, the presence of one or more tight junctions (zonula occludens) and the presence of well-organized zonula adherens. The presence of actin and nonmuscle myosin in EE cells suggests that the trans-endocardial permeability may be mediated by contraction or retraction of EE cells by phosphorylation of actin-binding proteins, such as vinculin and a-catenin, but also by activation of actin-myosin interactions.

Endothelial cells are interconnected via sets of binding proteins which form occludent, tight junctions (zonula occludens), adherens junctions (zonula adherens) and communicative junctions (gap junctions), whereas gap junctions form transmembrane channels between the adjacent cells, tight junctions and adherens junctions form pericellular zipper-like structures along the cell border through their transmembrane homophilic adhesion (Schema 1).

Tight junctions are located at the luminal side of the lateral membranes between adjacent EE cells and represent about 20% of total junctional complexes present in endothelial cells. They are composed of claudins, occludin, and junctional adhesion molecules. These junctions are mostly responsible for the restriction of the passage of water, electrolytes and small molecules through the endothelium. The role of tight junctions in regulating endothelial permeability remains incompletely understood. The expression level of occluding was found to correlate with enhanced endothelial barrier properties. Thus, occluding through its interaction with ZO-1 and the actin cytoskeleton stabilizes tight junctions [13].

Adherens junctions lie just below the tight junctions and they secure the junction between adjacent cells. Within the gap about 15 - 20 nm between the two cells, there is a cell membrane glycoproteincadherin. The cadherins from adjacent cells interact to 'zipper' up the two cells together. Within the cells, actin filaments (microfilaments) achieve their adhesive role and tend to be arranged circumferentially around the cell, into what is called a 'marginal' band. This marginal band may contract and also deform the shape of cells held together. Thus, adherens junctions position cells within the endothelium. The stability of these junctions depends on the concentration of calcium ions, in the absence of which the junctions break [14].

Numerous communicating gap junctions, 2 - 3 [micro]m in size, interconnect the cytoplasm of multiple cells. Permeable junctions in the EE provide the characteristics of a 'functional syncytium' because they coordinate the function of a set of cells. Connexins are gap junction proteins and represent a protein family with high homology in their amino acid sequences. Connexins aggregate to form hexamers. Hexamers of two adjacent cells form a connexon gap junction. Permeable junctions allow the passage of molecules up to 1 kDa in size and a rapid exchange of information in the form of molecules of low molecular weight, secondary messengers, [Ca.sup.2+] and inositol triphosphatate between the adjoining cells [12, 15].

Connexins have several regulatory sites on the cytoplasmic side that regulate the state of permeable junctions. The functional state of permeable junctions is influenced by voltage, pH [Ca.sup.2+] ions, calmodulin, phosphorylation, and G proteins [16]. The binding of a ligand to the receptor at one EE cell leads to cascade signalization amplifying cellular response. Alterations in both the amount and cellular distribution of gap junctions have been reported in many types of cardiac disease, and it has been suggested that these changes may cause arrhythmias and/or sudden cardiac death. Cardiac diseases are often associated with a reduced and/or heterogeneous expression of connexins. In atherosclerosis, expression of Cx37, Cx40, and Cx43 varies throughout the progression of the disease and connexins play different roles in plaque development [17].

Electrophysiological Characteristics of the Blood-Heart Barrier

The EE cells are electrically highly active, similarly to the brain capillary endothelial cells. Electrophysiological studies have demonstrated the existence of a large number of membrane ion channels: inward rectifier [K.sup.+] channels ([I.sub.kr]), [Ca.sup.2+] dependent [K.sup.+] channels ([K.sub.Ca]), voltage-dependent [Cl.sup.-] channels, the volume activated Cl - channels, stretch activated cation channels and a carrier-mediated transport, [Na.sup.+][K.sup.+] adenosine tripzhosphatase (ATPase) [18]. The asymmetric nature of the luminal compared to abluminal localization of ion channels and [Na.sup.+][K.sup.+] ATPase suggests that the net transcellular ionic transport from the blood to the cardiomyocyte interstitium occurs via a passive diffusion through the ion channels and through the active, carrier dependent transport.

Transendocardial electrical resistance in the endothelial cells of the right ventricle is two to five times higher than in other endothelial membranes (6-25 ohm/[cm.sup.2]) [19]. This is consistent with the assumption that EE functions as an active barrier between the circulating blood and cardiomyocyte interstitium. By combining the applicable electrophysiological techniques, Western blot and realtime polymerase chain reaction, in the EE cell culture we depicted a significant presence of [Na.sup.+][K.sup.+]-ATPase, predominantly of alpha-1-type, typically associated with the luminal membrane of EE cells [20, 21]. This asymmetric configuration can explain the net [Na.sup.+] transport from the heart into the blood and [K.sup.+] transport from the blood to the heart. A lower interstitial sodium level in the heart is more favorable for ensuring electrical stability, while a higher level of interstitial sodium causes the necessary excitability.The atrial volume reflex arc is an important contributor to the maintenance of bodily homeostasis, primarily responding to blood volume changes. Atrial volume receptors located in the endocardium of the atrial wall undergo mechanical deformation as blood is returned to the atria of the heart. The mechanosensitive channel(s) responsible for regulating plasma are the transient receptor potential (TRP) channel family members TRPC1 and TRPV4, expressed in sensory nerve endings in the atrial endocardium [22].

Conclusion

All endocardial endothelial cells act together in the organization of the endocardium as a functional syncytium to achieve a complex, well-organized, auto- and paracrine mediated physicochemical barrier between the circulating blood and subjacent heart tissue. Endothelial cells are able to dynamically regulate paracellular and transcellular transport of dissolved particles and water.

Paracellular permeability is determined by complex structures of junctions and intercellular adhesive strengths balanced with the contra-adhesive properties generated by the molecular mechanism of actin-myosin. The intact endothelial barrier limits the transport primarily by closing the interendothelial junctions. Through their receptor sites, integrins are related to the extracellular matrix, thus contributing to the stabilization of the barrier function of sealed intercellular spaces. While binding to their receptors, inflammatory mediators, thrombin, bradykinin, histamine, vascular endothelial growth factor and others, disturb the organization of interendothelial junctions and the integrin-extracellular matrix complex formation, thus creating the junctions that constitute the barrier.

Numerous diseases of the cardiovascular system can be a consequence but also the cause of endocardial endothelial dysfunction. Selective damage to the endocardial endothelium and subendocardium occurs in arrhythmia, atrial fibrillation, ischemia/reperfusion injury, cardiac hypertrophy and heart failure. Typical lesions of endocardial and microvascular endothelium have also been described in sepsis, myocardial infarction, inflammation, thrombosis, and in hypertensive patients. The result of endothelial dysfunction is the weakening of the endothelial barrier regulation and the electrolyte imbalance of the subendocardial interstitium.

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Sonja SMILJIC (1), Sladana SAVIC (2), Zvezdan MILANOVIC (1) and Goran GRUJIC (3)

Medical Faculty, Pristina, Kosovska Mitrovica

Department of Physiology (1)

Department of Histology (2)

General Hospital Pozarevac, Department of Cardiology (3)

Corresponding Author: Prof, dr Sonja Smiljic, Univerzitet u Pristini, Kosovska Mitrovica, Medicinski fakultet, Institut za fiziologiju, 38220 Kosovska Mitrovica, Anri Dinana bb, E-mail: sonja.smiljic@med.pr.ac.rs

Rad je primljen 6. V 2017.

Recenziran 28. IX 2017.

Prihvacen za stampu 12. X 2017.

BIBLID.0025-8105:(2018):LXXI:1-2:60-64.

https://doi.org/10.2298/MPNS1802060S
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Title Annotation:Seminar for physicians Seminar za lekare u praksi
Author:Smiljic, Sonja; Savic, Sladana; Milanovic, Zvezdan; Grujic, Goran
Publication:Medicinski Pregled
Date:Jan 1, 2018
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