Right Atrial Morphology in Coronary Heart Disease and Degenerative Aortic Valve Stenosis. Summary of the Doctoral Thesis

One of the main forms of cardiovascular diseases is coronary heart disease (CHD) but degenerative aortic valve (AoV) stenosis is the most frequent native valve disease. Both CHD and degenerative AoV stenosis have common risk factors such as age, high blood cholesterol, diabetes, smoking, high blood pressure, inflammation, and metabolic syndrome. Not only risk factors, but also pathophysiological changes, especially in the early stages of degenerative aortic valve stenosis, are similar to atherosclerosis - endothelial damage, lipid deposition, focal sclerosis, inflammatory cell infiltration, cytokine release and calcification. However, these conditions are not always observed at the same time. This confirms the existence of risk and pathogenesis factors specific to each disease. Although these heart diseases have been known for a long time and are intensively studied, there is still a lack of reliable markers that could help predict disease progression, the need for further surgery and mortality, therefore the pathophysiological processes involved in disease pathogenesis should be re-evaluated. Tissue changes in these diseases are complex and include cell death, cardiac innervation, tissue ischemia, regulators of metabolism and homeostasis, markers of inflammation and anti-inflammation, and other changes that are still not fully understood. Aim of the study: to determine the prevalence of markers of apoptosis, homeostasis regulating factors, innervation, ischemia and inflammation in right atrial tissue in cases of coronary heart disease and degenerative aortic valve stenosis. The tissue material used in the study – fragments of the right atrial appendage collected during elective open heart surgeries. A total of 36 patients with acquired heart diseases were included in the study – 24 patients with coronary heart disease and 12 patients with degenerative aortic valve stenosis. Samples of right atrial tissue from 5 patients with congenital heart disease operated at an early age were used as the study control group. Tissues were stained with hematoxylin and eosin for routine light microscopy, treated with the biotin-streptovidine method for immunohistochemical detection of tissue markers and by the TUNEL method for the detection of apoptotic cells. The following markers were identified in right atrial tissue by immunohistochemistry: atrial natriuretic peptide (ANUP), PGP 9.5- containing innervation, vascular endothelial growth factor (VEGF), chromogranin A (ChgA), endothelin 1 (ET-1), interleukin 1α (Il-1α ), interleukin 10 (II-10), β defensins 2, 3 and 4 (βD2, βD3 and βD4, respectively). Right atrial tissue in both CHD and degenerative AoV stenosis is characterized by non-specific degenerative morphological changes – pronounced vacuolization as well as changes in the shape and size of cardiomyocytes and their nuclei. In addition, these patients have a high proportion of apoptotic cardiomyocytes. Although there were no significant lesions in the coronary arteries in patients with AoV stenosis, connective tissue ingrowth and vascular sclerosis were observed in some patients in both groups. In the case of CHD and degenerative AoV stenosis, activation of the right atrial endocardial endothelial cells occurs, characterized by a change of shape from flat to cubic and rich release of ChgA, ET-1, Il-1α, Il-10, βD2 and βD3. Patients with CHD and AoV stenosis in the right atrial tissue had statistically significant higher numbers of ANUP-positive cardiomyocytes, all types of IL-10 positive cells and βD2 and βD3-positive endocardial endothelial cells, but fewer ChgA-positive cells than controls or patients with congenital heart disease. Thus, in both cases of acquired heart disease, an anti-inflammatory response prevails in the right atrial tissue, but increased activity of the neuroendocrine system is more common in patients with congenital heart disease at an early age. Although some tendencies were observed, for example, in the CHD group, there were slightly more VEGF, ET-1, Il-1α positive endocardial endothelial cells, Il-10 positive cardiomyocytes, connective tissue and endothelial cells, but in AoV stenosis group, there were slightly more ChgA-positive endocardial endothelial cells, however, these differences did not reach statistical significance. The most striking finding in our study was the rich expression of antimicrobial peptides, such as human β defensins 2 and 3, in the right atrial tissues in patients with CHD, degenerative AoV stenosis and congenital heart disease.

Homeostasis Regulating Factors, Innervation, Ischemia and Inflammatory Markers in the Right Atrial Tissue from Patients with Degenerative Aortic Valve Stenosis and Coronary Heart Disease

Both coronary heart disease (CHD) and degenerative aortic valve (AoV) stenosis have common risk factors, such as age, high blood cholesterol, diabetes, smoking, high blood pressure, inflammation, and metabolic syndrome. However, these diseases are not always observed together, confirming the existence of risk and pathogenesis factors specific to each disease. The aim of this study was to identify presence and distribution of common and different homeostasis regulating factors, innervation, ischemia and inflammatory markers in the right atrial tissue from patients with degenerative AoV stenosis and CHD. During elective cardiac surgery, right atrial tissue fragments were taken from 20 patients with CHD and from 9 patients with degenerative AoV stenosis. All tissue fragments were stained for immunohistochemical detection of protein-gene peptide 9.5 (PGP 9.5), atrial natriuretic peptide (ANUP), vascular endothelial growth factor (VEGF), chromogranin A, endothelin, interleukin 1 and 10 (Il-1 and Il-10) and β defensins 2, and 3 (βD2 and βD3). For the quantification of structures, a semi-quantitative counting method was used. Mostly numerous Il-10 positive cardiomyocytes and epi-/endocardial endothelial cells were detected in all specimens taken from patients with CHD, and statistically more than in specimens taken from patients with degenerative AoV disease (p = 0.007 and p = 0.016). Also, the number of βD3 positive cardiomyocytes was higher in the coronary heart disease group (p = 0.026). All other tested markers such as PGP 9.5, ANUP, VEGF, endothelin, chromogranin A, Il-1 and βD2 showed similar expression in both groups. Increased production of ANUP in right atrial tissue characterises both CHD and degenerative AoV stenosis. Production of ChgA in right atrial endocardial endothelial cells might represent regulation of sympathetic activity as a compensatory homeostatic response. Increased PGP 9.5-containing innervation is characteristic in patients with degenerative AoV disease and secondary mitral insufficiency. A stable increase of VEGF and variations of endothelin without statistically significant difference suggest influence of ischemia on the local vascular blood supply. Decreased production of Il-1α together with moderate to rich production of Il-10, βD2, and βD3 indicates the dominance of the local immune system over inflammation.

Angiotensin II induces apoptosis of human right and left ventricular endocardial endothelial cells by activating the AT2 receptor

Endocardial endothelial cells (EECs) form a monolayer lining the ventricular cavities. Studies from our laboratory and the literature have shown differences between EECs isolated from the right and left ventricles (EECRs and EECLs, respectively). Angiotensin II (Ang II) was shown to induce apoptosis of different cell types mainly via AT1 receptor activation. In this study, we verified whether Ang II induces apoptosis of human EECRs and EECLs (hEECRs and hEECLs, respectively) and via which type of receptor. Using the annexin V labeling and in situ TUNEL assays, our results showed that Ang II induced apoptosis of both hEECRs and hEECLs in a concentration-dependent manner. Our results using specific AT1 and AT2 receptor antagonists showed that the Ang-II-induced apoptosis in both hEECRs and hEECLs is mediated mainly via the AT2 receptor. However, AT1 receptor blockade partially prevented Ang-II-induced apoptosis, particularly in hEECRs. Hence, our results suggest that mainly AT2 receptors mediate Ang-II-induced apoptosis of hEECRs and hEECLs. The damage of EECs would affect their function as a physical barrier between the blood and cardiomyocytes, thus affecting cardiomyocyte functions.

Endocardial endothelium as a blood-heart barrier

Introduction. Endocardial endothelium is formed from a single layer of closely related cells with complex interrelationships and extensive overlap at the junctional edges. Morphological characteristics of blood-heart barrier. Endocardium is composed of three layers: endocardial endothelium, subendothelial loose connective tissue and subendocardium. The fibrous component of the subendothelium consists of small amount of collagen and elastic fibers. Several cell types are present in subendocardium: telocytes, fibroblasts and nerve endings. Intercellular bonds between the endocardial endothelial cells. Endocardial endothelial cells are attached to one another via sets of binding proteins forming solid, adherent and communicating connections. Communicating connections form transmembrane channels between the neighboring cells, while solid and adherent connections form pericellular structures like stitches. The maintenance of the presumed transendocardial electrochemical potential difference provides a high gradient for certain ions as well as a selective boundary barrier, basal lamina, preventing ionic leakage. The negatively charged glycocalyx also modulates endothelial permeability. Electrophysiological characteristics of heart-blood barrier. Electrophysiological studies have shown the existence of a large number of membrane ion channels in the endocardial endothelial cells: inward rectifying K+ channels, Ca2+ dependent K+channels, voltage-dependent Cl-channels, volume-activated Cl-channels, stretch-activated cation channels and one carrier mediated transport mechanism - Na+K+adenosine triphosphatase. Conclusion. Numerous diseases of the cardiovascular system may be a consequence, but also the cause of the endocardial endothelium dysfunction. Selective damage to the endocardial endothelium and subendocardium is found in arrhythmia, atrial fibrillation, ischemia/reperfusion injury and heart failure. Typical lesions of endocardial and microvascular endothelium have also been described in sepsis, myocardial infarction, inflammation and thrombosis. The result of endothelial dysfunction is the weakening of the endothelial barrier regulation and electrolyte imbalance of the subendocardial interstitium.

Neuropeptide Y and its receptors in ventricular endocardial endothelial cells

Endocardial endothelial cells (EECs) constitute an important component of the heart. These cells form a monolayer that covers the cavities of the right (EECRs) and left (EECLs) ventricles. They play an important role in cardiac excitation–contraction coupling via their secretion of cardioactive factors such as neuropeptide Y (NPY). They also contribute to cardiac pathology such as arrhythmia, hypertrophy, and heart failure. Differences between EECRs and EECLs contribute to tuning of circulating factors at the entry and exit of the ventricles. NPY, via activation of its receptors, modulates the excitation–secretion coupling of EECs, thus, indirectly modulating cardiac function and remodeling.

Difference in the response to angiotensin II between left and right ventricular endocardial endothelial cells

Previous studies focused on the right ventricular endocardial endothelial cells (EECRs) and showed that angiotensin II (Ang II) induced increase in cytosolic and nuclear calcium via AT1 receptor activation. In the present study, we verified whether the response of left EECs (EECLs) to Ang II is different than that of EECRs. Our results showed that the EC50 of the Ang II-induced increase of cytosolic and nuclear calcium in EECLs was 10× higher (around 2 × 10−13 mol/L) than in EECRs (around 8 × 10−12 mol/L). The densities of both AT1 and AT2 receptors were also higher in EECLs than those previously reported in EECRs. The effect of Ang II was mediated in both cell types via the activation of AT1 receptors. Treatment with Ang II induced a significant increase of cytosolic and nuclear AT1 receptors in EECRs, whereas the opposite was found in EECLs. In both cell types, there was a transient increase of cytosolic and nuclear AT2 receptors following the Ang II treatment. In conclusion, our results showed that both AT1 and AT2 receptors densities are higher in both EECLs compared to what was reported in EECRs. The higher density of AT1 receptors in EECLs compared to REECs may explain, in part, the higher sensitivity of EECLs to Ang II.