Inflammation and vascular remodeling in COVID-19 hearts

Abstract

A wide range of cardiac symptoms have been observed in COVID-19 patients, often significantly influencing the clinical outcome. While the pathophysiology of pulmonary COVID-19 manifestation has been substantially unraveled, the underlying pathomechanisms of cardiac involvement in COVID-19 are largely unknown. In this multicentre study, we performed a comprehensive analysis of heart samples from 24 autopsies with confirmed SARS-CoV-2 infection and compared them to samples of age-matched Influenza H1N1 A (n = 16), lymphocytic non-influenza myocarditis cases (n = 8), and non-inflamed heart tissue (n = 9). We employed conventional histopathology, multiplexed immunohistochemistry (MPX), microvascular corrosion casting, scanning electron microscopy, X-ray phase-contrast tomography using synchrotron radiation, and direct multiplexed measurements of gene expression, to assess morphological and molecular changes holistically. Based on histopathology, none of the COVID-19 samples fulfilled the established diagnostic criteria of viral myocarditis. However, quantification via MPX showed a significant increase in perivascular CD11b/TIE2 + -macrophages in COVID-19 over time, which was not observed in influenza or non-SARS-CoV-2 viral myocarditis patients. Ultrastructurally, a significant increase in intussusceptive angiogenesis as well as multifocal thrombi, inapparent in conventional morphological analysis, could be demonstrated. In line with this, on a molecular level, COVID-19 hearts displayed a distinct expression pattern of genes primarily coding for factors involved in angiogenesis and epithelial-mesenchymal transition (EMT), changes not seen in any of the other patient groups. We conclude that cardiac involvement in COVID-19 is an angiocentric macrophage-driven inflammatory process, distinct from classical anti-viral inflammatory responses, and substantially underappreciated by conventional histopathologic analysis. For the first time, we have observed intussusceptive angiogenesis in cardiac tissue, which we previously identified as the linchpin of vascular remodeling in COVID-19 pneumonia, as a pathognomic sign in affected hearts. Moreover, we identified CD11b + /TIE2 + macrophages as the drivers of intussusceptive angiogenesis and set forward a putative model for the molecular regulation of vascular alterations.

Keywords: Acute heart failure; Angiogenesis; CD11b; COVID-19; Coronavirus disease 2019; Heart; Intussusception; Intussusceptive angiogenesis; Macrophages; TIE2.

Fig. 1

A Histological comparison of COVID-19, influenza, and lymphocytic non-influenza myocarditis to non-infected controls. The myocardium of a COVID-19 patient shows no inflammatory infiltrate or cardiomyocyte necrosis with minor interstitial fibrosis (red arrowheads) and minor hypertrophy (COVID-19 patient ID 3). Myocardium of an influenza patient (Influenza patient ID 5) with minor interstitial fibrosis, moderate hypertrophy, and no inflammatory infiltrate or cardiomyocyte necrosis, whereas the myocardium in lymphocytic non-influenza myocarditis (Myocarditis patient ID 5) revealed dense infiltration of the inflammatory cells with single-cell necrosis (black arrowhead). Myocardium of a control patient (Control patient ID 8) displayed no inflammatory infiltrate, necrosis, fibrosis or hypertrophy, H&E staining, Magnification 100x, scale bar 100 µm. B Scanning electron micrographs of control heart tissue (left), COVID-19 (center), and lymphocytic myocarditis (right). In COVID-19 heart tissue (center), heart muscle fibers (myo) with slight hypertrophy are surrounded by a meshwork of collagenous fibers (black arrowheads). The orthogonal cardiac muscle orientation seems to be altered compared to the parallel organizations of myocardial strands (myo) in non-infected control heart tissue (left). The myocardial morphology in lymphocytic non-influenza myocarditis (right) is severely disturbed with pronounced edema, sporadic necrosis, and extensive lymphocytic infiltrates (yellow arrowheads), scale bars: 30 µm

Fig. 2

A–D MPX staining of cardiac tissue depicting CD68 + macrophages in orange, CD4 + T helper cells in green, CD8 + cytotoxic T cells in yellow, and CD20 + B-cells in magenta. All infected hearts (COVID-19, influenza, and lymphocytic non-influenza myocarditis) displayed a prominent infiltrate of CD68 + macrophages. While COVID-19 A (COVID-19 patient ID 24) and influenza, B (Influenza patient ID 9) hearts showed nearly absent lymphocytic infiltrate, lymphocytic non-influenza myocarditis, C (Myocarditis patient ID 5) was characterized by a mixed, T-cell dominated infiltrate. Non-infected control hearts, D (Control patient ID 1) showed markedly less inflammatory cells with a mixed population of macrophages and predominant t-cells and only scarce B-cells. Magnification 400x. Scale bars = 100 µm. E Histogram of the inflammatory cell infiltrates (CD20, CD4, CD68, CD8). Cell counts are normalized to cells per mm² myocardial tissue. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Macrophage expression of CD11b and TIE2. A Multiplex immunohistochemistry depicts a diffuse infiltration of CD11b⁺ macrophages (red) in the perivascular connective tissue in COVID-19 hearts (COVID-19 patient ID 17), (cardiomyocytes green, nuclei blue), scale bar 100 µm. B Bar diagram showing relative infiltration of CD11b⁺ inflammatory cells in non-infected control (Ctrl), influenza (Inf), COVID-19 (CoV), and lymphocytic non-influenza myocarditis (Myo) heart specimens morphometrically assessed by multiplex immunohistochemistry (MPX). Cell counts are normalized to cells per mm² tissue. C Immunohistochemical staining against TIE2 demonstrates the perivascular localization of TIE2⁺ cells (red arrowheads) in the myocardium of a COVID-19 patient (COVID-19 patient ID 17), scale bar: 10 µm. D Bar diagram showing the infiltration of Tie-2⁺ inflammatory cells in non-infected control (Ctrl), influenza (Inf), COVID-19 (CoV), and lymphocytic non-influenza myocarditis (Myo) heart specimens. Cell counts are normalized to cells per mm² myocardial tissue. Due to the small sample size of lymphocytic non-influenza myocarditis and a high variance among the samples, no statistical tests for significance were carried out. COVID-19 specimens were subdivided into two cohorts of cases with a hospitalization time < 10d and > 10d. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Differential regulation of mRNA expression in the cardiac tissue of COVID-19, influenza, and lymphocytic non-influenza myocarditis cases assessed by multiplex NanoString nCounter analysis system. The boxplots show the expression levels of representing genes of inflammation, hypoxia, angiogenesis, monocyte recruitment, and cell adhesion. Boxplots showing normalized log2 counts of mRNA expression and standard error of the mean, whiskers indicate outliers. Significance was evaluated with Benjamini–Hochberg correction. *FDR < 0.05, **FDR < 0.01, ***FDR < 0.001

Fig. 5

Visualization of ultrastructurally detectable thrombi (uTh) in COVID-19 hearts. A H&E staining of a thrombus in a smaller blood vessel in a field of interstitial fibrosis. B Immunohistochemical staining against activated fibrin displayed the formation of thrombus in a larger blood vessel. C Occasionally, small intracapillary megakaryocytes were observed in COVID-19 autopsy tissue, magnification 400× (COVID-19 patient ID 9). D, E Scanning electron micrograph of microvascular corrosion casting depicting numerous irregularly dilated and blind-ending vessels with vanishing microvascular hierarchy and micro-extravasation (black arrowheads) indicative for microangiopathy in COVID-19 heart tissue. Cardiac involvement of COVID-19 demonstrates caliber changes with dilated segments and focal vasoconstrictions (red arrowheads). The expansion of vascular plexus by intussusception (yellow arrowheads) is distinctly occurring in the dilated vessel segments, preferably on sites of vessel branching. Scale bars 100 µm. F Quantification of visible microthrombi (indicated by premature obliteration of the capillary network (approximate diameter 1–3 µm)) and intussusceptive neoangiogenesis (indicated by the formation of intussusceptive pillars) in COVID-19 and lymphocytic non-influenza myocarditis compared to healthy control tissue. G Correlation between the presence of uTh formation and the number of intussusceptive pillar formation in COVID-19 and lymphocytic non-influenza myocarditis compared to non-infected control tissue

Fig. 6

Assessment of the cardiac microvasculature by microvascular corrosion casting and X-ray phase-contrast tomography. Scanning electron micrographs of microvascular corrosion casts revealed in COVID-19 hearts A a distorted microvascular architecture with dilated segments and blind-ending stocks (black arrowheads), whereas the vascular architecture of control heart tissue, B displayed a regular hierarchical organization (approximate vessel diameter 1–3 µm). C Microvascular corrosion casting exposed an irregularly shaped and tortuous microvascular architecture with numerous tiny holes, scale bar 20 µm. D Scanning electron micrograph of COVID-19 cardiac vascular plexus highlights the confluent expansion of transluminal intussusceptive pillars (black arrowheads) at a branching point, often seen as doublets and triplets, scale bar 5 µm. E Volume rendering of a tomographic reconstruction obtained by synchrotron-radiation-based X-ray phase-contrast tomography highlighting the mild interstitial fibrosis (orange) in COVID-19 heart tissue. In the magnification of the presented 2D-slice (marked by a red rectangle) a nucleus of an endothelial cell and an intraluminal pillar (black arrowheads) are visible. F Volume segmentation of microvasculature (depicted in orange) in phase-contrast synchrotron-radiation-based-X-ray tomographs demonstrate the altered microvascular architecture (Arrowheads) in COVID-19 hearts compared to the parallel alignment of coronary plexus in control hearts. The reconstructed dataset shown in F has been recorded at 167 mm voxel size. A cube of 350 µm side length is shown

Fig. 7

Functional gene expression analysis of COVID-19, influenza, and lymphocytic non-influenza myocarditis heart tissues. A Functional Pathway analysis via gene set enrichment analysis against the Gene Ontology aspect biological functions highlights the differential functional gene expression in the heart tissue of COVID-19 and influenza patients. The activation of biological functions in cardiac injury patterns compared to healthy controls was predicted for each sample. Color indicates up- (red) and down (blue)-regulation; circle size depicts FDR. Only significantly up-or down-regulated pathways are shown. B Spider-Plot depicting the enrichment of biological functions from Gene Ontology based on gene expression data of COVID-19 and influenza heart samples as compared with expression in non-infected control specimen. The y-axis shows the normalized enrichment scores (NES) on a scale from 0 to 2. C Venn diagram of statistically differentially expressed genes of COVID-19 and influenza heart samples as compared with expression in controls in both disease groups (Student’s t test, controlled for the familywise error rate with a Benjamini–Hochberg false discovery rate threshold of 0.05). Up-regulation and down-regulation of genes are indicated by colored arrowheads suffixed to the gene symbols (green denotes upregulation, red denotes down-regulation). Numbers given are the total of differentially regulated genes, displayed are the top 10 up- or down-regulated genes. Note that there were no differentially expressed genes for lymphocytic non-influenza myocarditis

Fig. 8

Visualization of the hypothesis of CD11b + /TIE2 + monocytes/macrophages recruitment and incorporation, and intussusceptive angiogenesis in the cardiac vasculature in COVID-19. SARS-CoV-2-related endothelial dysfunction results in thrombotic microangiopathy in cardiac capillaries and tissue hypoxia. Endothelial cells induce the recruitment of monocytes/macrophages to the site of injury by upregulation of adhesion-molecules and activation of SDF-1/CXCR4 signaling. TIE2 + monocytes/macrophages are activated by increased levels of angiopoietin 1 and adhere locally in response to angiopoietin 2 to endothelial cells. Formation of an intussusceptive pillar is achieved by intraluminal stretching of endothelial cells under the help of adherent TIE2 + monocytes/macrophages resulting in the division of a single capillary altering the cardiac microvasculature