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Abstract
Context: Researchers throughout the world devote enormous efforts to reveal the peculiarities of the pathogenesis of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, however, it continues to surprise and cause the death of millions of people. Aims: This article aims to study the molecular mechanisms provoked by SARS-CoV-2, the virus-induced changes in Angiotensin-converting enzyme 2 (ACE2) functionality, in the vascular homeostasis through CD34 expression, B-cell immunity through the expression of CD20 and CD79α, and adhesion molecules through E-cadherin. Settings and Design: This was a prospective, descriptive, and observational study. Methods and Material: A total of 15 autopsies of patients deceased by COVID-19 infection, confirmed by PCR, were performed. The lungs of all patients were examined histologically and immunohistochemically for ACE2, E-cadherin, CD34, CD20, and CD79α. Results: Immunohistological analysis showed increased ACE2 expression in all lung autopsy material affected by COVID-19 infection and we found a higher intensity of ACE2 expression than that of a healthy lung. CD20 examination reveals total deficiency of B-cells in the pulmonary parenchyma and CD79α is also absent. E-Cadherin is not expressed in the basal cellular sections where the contact elements are missing. CD34 demonstrates a desquamation of the endothelial cells, which indicates a direct damage of the vascular walls. Conclusions: We found that patients who died after severe COVID-19 had high immune deficiency and impaired intercellular communication in the parenchyma and endothelium of lung tissue, leading to severe thromboembolic complications in patients with multiple diseases.
Keywords: Angiotensin-converting enzyme 2, COVID-19, respiratory diseases, SARS-CoV-2
How to cite this article: Introduction 
The role of ACE2 in establishing the connection between SARS-CoV-2 and its penetration in the cell has been ascertained.[1] Understanding the molecular mechanisms of ACE2 activity on cell signaling, on one hand as a main receptor of SARS-CoV-2 and on the other hand as part of the renin-angiotensin system, could provide guidance in comprehending and solving the observed clinical picture of severe respiratory, renal, and cardiac disabilities in patients with cardiac and metabolic problems after SARS-CoV-2 infection.[2]
To observe the effects connected with SARS-CoV-2 through ACE2 in different type of cells, we used various immunological markers as CD34 specific marker for endothelial cells, CD20 and CD79a for B-lymphocytes, and E-cadherin for airway epithelial layer in the lungs.
Subjects and Methods PCR-test confirming COVID-19
COVID-19 in vitro diagnostic kit detects SARS-CoV-2 (E gene and RdRp gene) RNA from infected patient's sample (such as sputum, nasopharyngeal swab, and oropharyngeal swab) through Real-Time Polymerase chain reaction (RT-PCR) AccuPower® SARS-CoV-2 RT-PCR Kit (Bioneer, Korea, South).
Autopsy procedures
All deceased patients have a positive PCR test proving that they were infected with COVID-19. This is a prospective study of 15 consecutive COVID-19 autopsies performed in St. George University Hospital, Plovdiv, Bulgaria. For a control, we used the lungs of a 40-year-old man who died after an accident.
Clinical data
Most patients are over 50 years of age with many accompanying diseases. Only one patient at the age of 61 did not report any concomitant condition. However, there was a patient under 40 years of age who had high blood pressure, left ventricular hypertrophy (LVH), and obesity (grade III). From the studied patients, the most common diseases among them are high blood pressure (hypertension), LVH (found in 13 out of 15 tested subjects), chronic ischemic heart disease (found in 10 patients), atherosclerosis (found in 10 patients), diabetes mellitus (found in 5 patients), and obesity (grade III) (found in 14 out of 15 tested patients).
Autopsy
For safety precautions 4 autopsies were partial, the cranium not being opened, the rest 11 autopsies were complete. Material was obtained from the lungs—from the central regions of the two lungs, as well as from the periphery. Four blocks for routine H.E. examination were processed. Small arteries were defined as < 1.0 mm; the term “medium-sized pulmonary vessels” was used for those larger than 1.0 mm, often clearly visible, unlike pulmonary emboli in the main pulmonary artery.
Histological examination
Autopsy material was fixed in 10% neutral buffered formalin for 24 to 48 h and submitted for standard processing with hematoxylin and eosin staining.
Immunohistochemistry
Immunohistochemical staining was performed on formalin fixed, paraffin-embedded 5-μm sections following citrate pH 6.0 antigen retrieval, endogenous biotin, and peroxidase block.
The lungs were tested immunohistochemically with ACE2; E-Cadherin to establish the changes of the vascular endothelium and alveolocytes; CD 34 for endothelial damage; CD20 and CD79α for presence of B-lymphocytes. Immunohistochemical (IHC) staining was performed by Autostainer Link 48 (Dako, Denmark). Images were visualized and captured with a digital camera mounted on a Nikon Eclipse 80i microscope using NIS-Elements Advanced Research Software version 4.13 (Nikon Instruments, Tokyo, Japan).
Results We performed immunohistochemical examination of ACE2, CD20, CD34, CD79α, and E-Cadherin in a control group of patients without lung disease, and necropsy lung tissue of patients with SARS-CoV-2. Macroscopically, the lungs were bilaterally enlarged, heavy (600–700 g), with greatly reduced elasticity and increased density. Their surface was homogeneous, with a brownish-reddish color and many fibrin thrombi in medium-sized pulmonary vessels. Intraparenchymal hemorrhages were observed. Histopathological examination of the pulmonary system revealed a spectrum of diffuse alveolar damage, presence of intra-alveolar fibrin, hyaline membranes, or loosely organized connective tissue in the alveolar septal walls. Airways and alveolar spaces contained large, reactive multinucleated cells.
Multiple fibrin thrombi were found in medium-sized pulmonary vessels without signs of any inflammation of the vascular wall, which indicates direct damage of endothelial cells [Figure 1]a. Small vessels (arterioles, capillaries, and venules) were not affected [Figure 1]b.
Figure 1: Hematoxylin and eosin-stained slide of paraffin-embedded tissue. (a) Lung tissue with medium-sized vessels (Control lung, H-E, 40×). (b) Fibrin thrombus in a pulmonary vessel (severe COVID-19, 10×)ACE2—In all lung autopsy material affected by SARS-CoV-2 infection, we found a higher intensity of ACE2 expression than that of a healthy lung. ACE2 is well-visualized in alveolocytes, macrophages, endothelial cells, and smooth muscle cells [Figure 2]a and [Figure 2]b.
Figure 2: (a) ACE2 expression in the control of vessels and lung, without accompanying diseases (20×). (b) ACE2 expression in vessels and lung cells after SARS-Cov 2 (10×). (c) Control specimen, basal, and intercellular contacts are preserved, E-Cadherin (40×). (d) Loss of E-Cadherin in the basal areas of the cells (light arrow) and storage of contact elements between the cells in the syncytial structures (dark arrow), (40×)E-Cadherin is not expressed in the basal cellular sections where the contact elements are missing. There is preservation of contact elements between the cells in the syncytial structures which are detached “an bloc” from the basal membranes [Figure 2]c. In the controls of normal lung tissue, the contact elements with the basal membranes are preserved, as well as the contacts between the cells themselves [Figure 2]d.
In lung controls, small groups of B-cells lymphocytes, positive for CD20, were observed peribronchially. Normal lung tissue was with focal accumulations of B-lymphocytes around bronchioles and blood vessels [Figure 3]a. CD20 examination reveals total deficiency of B-cells in the pulmonary parenchyma [Figure 3]b. CD79a expression is also absent [Figure 3]f.
Figure 3: (a) Focal accumulation of B lymphocytes. (b) CD20 complete deletion of B-cells, autopsy lung (10×). (c) Preserved endothelial cells in intact lung (Control lung, CD34, 40×). (d) CD34 demonstrates desquamation of endothelial cells from vessel walls (20×). (e) CD79× expression in peribronchial group B lymphocytes, lung control, (20×). (f) CD79× lack of expression in lung with COVID-19 (10×)Expression of CD34 demonstrated preserved endothelial cells in intact lung [Figure 3]c. Immunohistochemically, CD34 demonstrates a desquamation of the endothelial cells from the vascular walls, which indicates a direct damage of endothelial cells [Figure 1]d.
Discussion In this study, we aimed at establishing the causes, which lead to severe infection and death following COVID-19. Therefore, we traced the pathomorphological changes in the lungs immunohistochemically the expression of ACE2, epithelial E-cadherin, CD20, CD79α, and CD34 in patients with SARS-CoV-2 and multiple concomitant diseases (chronic ischemic heart disease, atherosclerosis, diabetes mellitus, and obesity).
The individual response to SARS-CoV-2 is known to be highly heterogenous. However, according to our autopsies, the picture is quite identical.[3]
The identified post-mortem findings in the lungs of COVID-19 patients reveal increased expression of ACE2, loss of epithelial cadherin, reduced to lack of expression of CD20 and CD79α, markers for an adaptive immune response from B-lymphocytes and IgM memory cells, increased expression of CD34 in desquamated endothelial cells in the vascular walls, and multiple fibrin thrombi in mid-sized pulmonary vessels lacking any inflammatory reaction in the vascular wall. The microvessels (arterioles, capillaries, and venules) were not affected by microthrombosis.
The changes, which are observed, are multifactorial since the majority of patients have multiple concomitant diseases, which have led to the imbalance in the renin-angiotensin-aldosterone (RAAS) system.[4] Enhanced Ang II expression leads to inflammation of the cardiac muscle involving the vascular endothelium.[5] In response to the RAAS (Ang II) activity, the expression of ACE2 increases; however, this increased expression facilitates the entry of SARS-CoV-2 in cells.[6]
ACE2 is expressed from various types of cells, such as epithelial, endothelial, smooth muscle, alveolar macrophages, stem, and hematopoietic cells.[7] All these cells are targets of the virus; therefore, they can be damaged or lysed following contact with the virus.
We used different immunohistochemical markers to visualize and characterize the pathological infractions in the lung after SARS-CoV-2 infection.
We established an increased ACE2 expression which is found in patients with diabetes type 1 and type 2, patients with hypertension taking ACE2 inhibitors, and patients taking Angiotensin receptor blockers (ARBs) that lead to increased regulation of ACE2.[8]
The analysis of the CD34 expression as a specific marker showed increased desquamation of endothelial cells in the walls of the blood vessels.
The damaged cells begin to release inflammatory molecules. The endothelial and epithelial cells, as well as the alveolar macrophages in the lungs, identify these molecules following a release of pro-inflammatory cytokines, chemokines, and macrophage inflammatory proteins.[9]
Endothelial cells respond to the impairment with structural damage (lack of synthetic activity, impaired barrier, and anti-thrombogenic function).[9]
The impaired endothelial function was confirmed by the multiple fibrine thrombi in mid-sized pulmonary vessels, but small vessels (arterioles, capillaries, and venules) were not involved. In the described autopsy cases, we did not find any inflammatory changes in either medium-sized vessels or small capillary-type vessels, except for desquamation of the endothelium, which is a sufficient factor to initiate thrombogenesis. These findings support the theory that multiplication of the virus in the endothelium leads to cell necrosis and triggers thrombotic complications, cellular damage associated molecular patterns, and cell necrosis being an immune response restricting viral replication.[10]
This study confirms the hypothesis on the risk of generalized arterial and venous thromboses in coronavirus infection. What is more, this research demonstrates that viral endothelial injury and endothelial dysfunction alone are enough to trigger thrombogenesis in both arteries and veins.[10]
Since ACE2 is also expressed by the lung epithelium, we used E-cadherin to check the extent of the barrier dysfunction.
Using an immunohistochemical method, we established the loss of E-cadherin and denudation of the epithelium of the airways. A similar loss is reported in E-cadherin knock-out model,[11] as well as in children with asthma,[12] in which eosinophilic inflammation, impaired barrier function, and congenital immune responses with the characteristics of asthma and severe allergies are present. In the knock-out model, the reduced E-cadherin expression is observed in alveolar epithelial type II cells, which are responsible for the production of pulmonary surfactant which reduces the surface pressure in the alveoli.[11] These cells are crucial for the repair of the injured alveoli by differentiating into alveolar epithelial type I cells.[11],[12]
These disorders are probably the causes for difficult breathing and the necessity for intubation in severe COVID-19 infection cases. In these findings, the loss of E-cadherin in the airway epithelium is not simply a consequence of the disease but also actively participates in COVID-19 pathogenesis of the viral infection itself.[11]
The immune system responds to the virus by generating specific antibodies to the viral antigens, which bind to them, and in some cases, block their binding to the ACE2 receptor.
These are produced by plasma cells, formed by the differentiation of B lymphocytes within the lymph nodes.[13],[14]
The lack of CD20 and CD79α found by us confirms the depletion of IgM memory in B-cells.[15],[16] The lack of IgM memory in cells reveals a lack of immune response against the spread of SARS-CoV-2 toward other organs and immune response against other infections.[14],[17] The B-cell activation leads to the production of antibodies specific for the virus (IgG, IgM, and s IgA) for the neutralization of the viral toxicity.[18]
The loss of CD20 and CD79a is indicative of a lack of adaptive and cellular immune response. In normal conditions, B-cells mediate a greater part of the T-cell cellular-dependent response of the antibodies.[18]
The peculiarities of the immune system in severe COVID-19 are discussed in several reports. Lymphopenia with a drastically reduced number of T-cells, B-cells, and natural killer cells is observed which is not the case with milder cases.[18]
One of the possible causes for B-lymphocyte reduction is that ACE2 is expressed on the hemopoietic stem cells and in small quantities in the differentiating hemopoietic cells in the bone marrow, which allows for the direct entry of the virus and the subsequent activation of necroptosis, apoptosis, or pyroptosis in them.[19]
The mechanisms through which COVID-19 causes apoptosis in the lymphocytes are still not established. However, it is considered that pyroptosis occurs due to the activation of the NLRP3 inflammasome. In patients with severe concomitant diseases, NLRP3 inflammasome is thought to be activated by the high Ang II levels, since it is the basis of the disease pathogenesis itself.[20] The activated NLRP3 inflammasome causes cellular death not only in the lymphocytes and immune cells but also in the pulmonary epithelial cells disrupting the intercellular bonds, and in the blood vessels, it disrupts the integrity of the endothelium, gas exchange, and blood flow, causing clot formation observed in the performed biopsies of deceased patients following a severe COVID-19 infection.[21]
Conclusions We found that patients who died after severe COVID-19 were with high immune deficiency and impaired intercellular communication in the parenchyma and endothelium of the pulmonary tissue leading to severe thromboembolic complications in patients with multiple diseases.
Further research is necessary to identify the causes leading to immune system failure, a thorough study on the molecular level and drawing more detailed conclusions needs to be done.
Acknowledgments
The authors thank the Ministry of Education and Science of Bulgaria—National Program “Young Scientists and PhD Students” 2021 for financial support of the research and their publication.
Financial support and sponsorship
Project КP-06-DК1/6 - 29.03.2021”COVID-19 HUB - Information, Innovations and Implementation of Integrative Research Activities”, National Scientific Fund, Ministry of Education.
Conflicts of interest
There are no conflicts of interest.
References 
Correspondence Address:
Mina Miroslavova Pencheva
15A VasilAprilov Bul., Plovdiv
Bulgaria
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_734_21
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