Morphological and functional changes in microvasculature and endomysium in human atrial myocardium with atrial fibrillation

This doctorial thesis was focused on morphological and functional changes in microvasculature and endomysium in human atrial myocardium with atrial fibrillation. Atrial fibrillation (AF) is one of the most common arrhythmias in the clinical practice and it is associated with an increase in mortality risk that is strongly related with old age. Its pathogenesis is still not sufficiently explored. One of the generally recognized factors contributing to the initiation and maintenance of atrial fibrillation is structural remodeling of the myocardium. Structural remodeling is reflected by changes that affect both atrial cardiomyocytes as well as endomysium. We analyzed atrial biopsies obtained from patients undergoing bypass or mitral valve surgery. The patients had a regular sinus rhythm or were suffering from AF. Immunohistochemistry was used to visualize collagen I, collagen III, elastin, desmin, smooth muscle actin and VEGF in the atrial samples.To detect capillaries UEA-lectin was used. For detection of different types of immune cells the following markers were detected immunohistochemically: CD45 as a pan- leukocyte marker, CD3 for T-lymphocytes, CD68 for monocyte/macrophages, mast cell tryptase for mast cells and DC-SIGN for immature dendritic cells. Our results document that in patients...Tato disertační práce byla zaměřena na morfologické a funkční změny kapilárního řečiště a endomysia v lidském atriálním myokardu při fibrilaci síní. Fibrilace síní (FS) je jednou z nejčastějších arytmií, se kterou se setkáváme v klinické praxi, je spojena se zvýšeným rizikem úmrtnosti a silně souvisí se stářím. Její patogeneze stále není dostatečně prozkoumána. Jeden ze známých faktorů přispívajících k udržení FS je strukturální přestavba myokardu. Strukturální remodelace se projevuje jednak změnami samotných síňových kardiomyocytů, ale také změnami v endomysiu. Studovali jsme síňové biopsie získané od pacientů kteří podstoupili aorto-koronární bypass nebo operaci mitrální chlopně. Pacienti měli pravidelný sinusový rytmus nebo fibrilaci síní. Imunohistochemie byla použita k vizualizaci kolagenu I, kolagenu III, elastinu, desminu, aktinu hladkého svalstva a VEGF ve vzorcích síní. Pro detekci kapilár byl použit UEA-lektin. Pro detekci různých typů imunitních buněk byly imunohistochemicky detekovány následující markery: CD45 jako marker panleukocytů, CD3 pro T-lymfocyty, CD68 pro monocyty/makrofágy, tryptáza z žírných buněk pro žírné buňky a DC-SIGN pro nezralé dendritické buňky. Naše výsledky dokumentují, že u pacientů podstupujících operaci na otevřeném srdci lze nalézt variabilní hladinu ECM...Ústav histologie a embryologie 1. LF UKInstitute of Histology and Embryology First Faculty of Medicine Charles UniversityFirst Faculty of Medicine1. lékařská fakult

Morphological and functional changes in microvasculature and endomysium in human atrial myocardium with atrial fibrillation

This doctorial thesis was focused on morphological and functional changes in microvasculature and endomysium in human atrial myocardium with atrial fibrillation. Atrial fibrillation (AF) is one of the most common arrhythmias in the clinical practice and it is associated with an increase in mortality risk that is strongly related with old age. Its pathogenesis is still not sufficiently explored. One of the generally recognized factors contributing to the initiation and maintenance of atrial fibrillation is structural remodeling of the myocardium. Structural remodeling is reflected by changes that affect both atrial cardiomyocytes as well as endomysium. We analyzed atrial biopsies obtained from patients undergoing bypass or mitral valve surgery. The patients had a regular sinus rhythm or were suffering from AF. Immunohistochemistry was used to visualize collagen I, collagen III, elastin, desmin, smooth muscle actin and VEGF in the atrial samples.To detect capillaries UEA-lectin was used. For detection of different types of immune cells the following markers were detected immunohistochemically: CD45 as a pan- leukocyte marker, CD3 for T-lymphocytes, CD68 for monocyte/macrophages, mast cell tryptase for mast cells and DC-SIGN for immature dendritic cells. Our results document that in patients..

Quantification of Analgesic and Anti-Inflammatory Lipid Mediators in Long-Term Cryopreserved and Freeze-Dried Preserved Human Amniotic Membrane

The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators—palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)—in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM’s analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography–tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3–5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts

Bioptic Study of Left and Right Atrial Interstitium in Cardiac Patients with and without Atrial Fibrillation: Interatrial but Not Rhythm-Based Differences.

One of the generally recognized factors contributing to the initiation and maintenance of atrial fibrillation (AF) is structural remodeling of the myocardium that affects both atrial cardiomyocytes as well as interstitium. The goal of this study was to characterize morphologically and functionally interstitium of atria in patients with AF or in sinus rhythm (SR) who were indicated to heart surgery. Patient population consisted of 46 subjects (19 with long-term persistent AF, and 27 in SR) undergoing coronary bypass or valve surgery. Peroperative bioptic samples of the left and the right atria were examined using immunohistochemistry to visualize and quantify collagen I, collagen III, elastin, desmin, smooth muscle actin, endothelium and Vascular Endothelial Growth Factor (VEGF). The content of interstitial elastin, collagen I, and collagen III in atrial tissue was similar in AF and SR groups. However, the right atrium was more than twofold more abundant in elastin as compared with the left atrium and similar difference was found for collagen I and III. The right atrium showed also higher VEGF expression and lower microvascular density as compared to the left atrium. No significant changes in atrial extracellular matrix fiber content, microvascular density and angiogenic signaling, attributable to AF, were found in this cohort of patients with structural heart disease. This finding suggests that interstitial fibrosis and other morphological changes in atrial tissue are rather linked to structural heart disease than to AF per se. Significant regional differences in interstitial structure between right and left atrium is a novel observation that deserves further investigation

Collagen III in the atrial myocardium.

<p>A-D: Immunohistochemical reaction shows collagen III in endomysial and partly also perimysial extracellular matrix. In atria of both patient groups (SR in A, B and AF in C, D) it is possible to detect higher (A, C) as well as lower (B, D) amount of collagen III-positive ECM. Immunoperoxidase reaction with DAB as a substrate (brown precipitate). No nuclear counterstaining. For all images scale bar = 50μm. (E) A graph showing the result of quantification of collagen III volume fraction (CIIIVF) in atrial myocardial samples. A comparison between different anatomical locations is shown: right appendage–RA (n = 37), left appendage–LA (n = 19), left atrium—LAt (n = 9), LA+LAt (n = 28).</p

Expression of VEGF in atrial myocardium from patients with sinus rhythm (SR) and atrial fibrillation (AF).

<p>A, B: Atrial samples from patients with SR. (A) A strong immunoreactivity for VEGF is localized to the capillaries, while cardiomyocytes display rather low level of VEGF expression. (B) High level of VEGF immunoreactivity in mesothelial cells and in adipocytes of epicardium. C, D: Atrial samples from patients with AF. (C) A strong immunoreactivity for VEGF is localized to mesothelium. There are VEGF-positive capillaries and moderately positive cardiomyocytes in the atrial myocardium. (D) A strong VEGF immunoreactivity in the myocardium (mainly cardiomyocytes) and in mesothelial cells. Scale bar in A-D = 50μm. (E) A graph showing a result of semiquantitative analysis of VEGF immunoreactivity in the atrial samples from all patients as described in Methods (score 0–3). A comparison between different structures from different anatomical locations is shown: RA–right appendage, LA–left appendage, LAt–left atrium.</p

Microvessel pericyte coverage in atria of patients with sinus rhythm (SR) and atrial fibrillation (AF).

<p>(A-D) Confocal images of human atria. (A) Immunodetection of desmin (green) shows its expression in cardiomyocytes. Capillaries labeled with UEA-lectin (red) are devoid of desmin immunoreactivity (arrowheads). Scale bar = 50μm. (B) Desmin is present in cardiomyocytes, while SMA-positive capillary pericyte (arrowhead) does not display desmin signal. Scale bar = 10μm. (C) A representative section of myocardium used for the quantitative analysis of microvessel pericyte coverage index. Blood vessel endothelium is labeled using UEA lectin (red) and pericytes are visualized using anti-SMA antibody (green). Scale bar = 100μm. (D) A high magnification image shows SMA-positive smooth muscle cells surrounding a small arteriole and also SMA-positive pericytes in association with the capillaries (arrowheads). Scale bar = 25μm. (A, B, D) Nuclei are stained with DAPI (blue). (E) A graph showing results of quantification of microvessel pericyte coverage index (MPI) in atrial samples of all patients as described in Methods. RA–right appendage (n = 8), LA–left appendage (n = 9), LAt–left atrium (n = 7), LA + LAt (n = 16).</p

Analysis of immune cell populations in atrial myocardium of patients with atrial fibrillation or sinus rhythm

<div><p>Background</p><p>Atrial fibrillation (AF) is the most common arrhythmia and despite obvious clinical importance remains its pathogenesis only partially explained. A relation between inflammation and AF has been suggested by findings of increased inflammatory markers in AF patients.</p><p>Objective</p><p>The goal of this study was to characterize morphologically and functionally CD45-positive inflammatory cell populations in atrial myocardium of patients with AF as compared to sinus rhythm (SR).</p><p>Methods</p><p>We examined 46 subjects (19 with AF, and 27 in SR) undergoing coronary bypass or valve surgery. Peroperative bioptic samples of the left and the right atrial tissue were examined using immunohistochemistry.</p><p>Results</p><p>The number of CD3+ T-lymphocytes and CD68-KP1+ cells were elevated in the left atrial myocardium of patients with AF compared to those in SR. Immune cell infiltration of LA was related to the rhythm, but not to age, body size, LA size, mitral regurgitation grade, type of surgery, systemic markers of inflammation or presence of diabetes or hypertension. Most of CD68-KP1+ cells corresponded to dendritic cell population based on their morphology and immunoreactivity for DC-SIGN. The numbers of mast cells and CD20+ B-lymphocytes did not differ between AF and SR patients. No foci of inflammation were detected in any sample.</p><p>Conclusions</p><p>An immunohistochemical analysis of samples from patients undergoing open heart surgery showed moderate and site-specific increase of inflammatory cells in the atrial myocardium of patients with AF compared to those in SR, with prevailing population of monocyte-macrophage lineage. These cells and their cytokine products may play a role in atrial remodeling and AF persistence.</p></div

CD3-positive T-lymphocytes and CD68-KP1-positive cells in the atrial myocardium.

<p>Sections of atrial myocardium from patients with atrial fibrillation showing a result of immunoperoxidase reaction for CD3 and CD68-KP1. A) CD3-positive T-lymphocytes localized among atrial cardiomyocytes and in the interstitial spaces (arrowheads). Scale bar = 50 μm. B) CD68-KP1-positive cells are found in the interstitium and have mainly elongated morphology (arrowheads). Scale bar = 50 μm. C) Frequency of CD3-positive T-lymphocytes in the atrial myocardium of patients with atrial fibrillation (AF) and sinus rhythm (SR). An average number of CD3-positive T-lymphocytes cells per square mm of cross-sectioned atrial myocardium is given +- SD. Right appendage–SR (n = 17), AF (n = 11); Left appendage—SR (n = 8), AF (n = 10); Left atrial free wall + left appendage—SR (n = 9), AF (n = 16). *—p<0,05 D) Frequency of CD68-KP1-positive cells in the atrial myocardium of patients with atrial fibrillation (AF) and sinus rhythm (SR). An average number of CD68-KP1-positive cells per square mm of cross-sectioned atrial myocardium is given +- SD. Right appendage–SR (n = 22), AF (n = 9); Left appendage—SR (n = 11), AF (n = 9); Left atrial free wall + left appendage—SR (n = 13), AF (n = 19). *—p<0,05</p

CD45-positive cells in the atrial myocardium.

<p>Sections of atrial wall from patients with sinus rhythm (A, B, D) and atrial fibrillation (C) showing a result of immunoperoxidase reaction for CD45. A) Epicardial and myocardial layer of atrial wall contains scattered as well as clustered (arrowhead) CD45-positive cells. Scale bar = 100μm. B) Endocardial layer with CD45-positive cells (arrowhead) next to the atrial lumen (L). Scale bar = 20μm. C) A section through the trabecular part of the atrial wall with CD45-positive cells in the myocardium (M) and endocardium (E). Scale bar = 50 μm. D) A detailed view on the atrial myocardium with CD45-positive cells having rather rounded (arrowhead) or elongated (arrow) cell shape. Scale bar 50 = μm. E) Frequency of CD45+ cells in the atrial myocardium of patients with atrial fibrillation (AF) and sinus rhythm (SR). An average number of CD45+ cells per square mm of cross-sectioned atrial myocardium is given +- SD. Right appendage–SR (n = 22), AF (n = 15); Left appendage—SR (n = 8), AF (n = 8); Left atrial free wall—SR (n = 4), AF (n = 8); Left atrial free wall + left appendage—SR (n = 12), AF (n = 16).</p