A wide range of cardiac symptoms have been observed in COVID-19 patients, often significantly influencing the clinical outcome. Current hypotheses include direct and indirect injury to the myocardium by viral infection. Cardiomyocytes express significant levels of angiotensin-converting enzyme 2 receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the lack of transmembrane serine protease 2, which cleaves spike (S) protein allowing this binding to take place, likely hampers direct cardiomyocyte infection. Another hypothesis implicates the systemic release of pro-inflammatory cytokines, which increase vascular wall permeability, leading to myocardial edema. The German authors in this multicenter autopsy study evaluated, for the first time, the molecular, morphological, and ultrastructural alterations in cardiac tissue of patients who died of COVID-19, influenza A virus subtype H1N1, and non-influenza lymphocytic (e.g., coxsackievirus) myocarditis using histology, immunohistochemistry, vascular corrosion casts, electron microscopy, and gene expression analysis. They proposed that a similar vascular tropism, subsequent endothelial damage, and marked alterations of the cardiac microvasculature characterize cardiac injury in COVID-19.
The authors pointed to variable findings of conventional heart histopathology in COVID-19 cases, ranging from typical lymphocytic myocarditis and thrombotic microangiopathy to the absence of significant lymphocytic infiltrates and myocyte damage. A slight increase in perivascular macrophages, often referred to as borderline myocarditis, was the most frequently reported morphological finding. Furthermore, the majority of cases did not meet the established Dallas criteria for myocarditis.
Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels. According to the current consensus, there are two types of angiogenesis: sprouting and splitting (intussusceptive) angiogenesis. Intussusceptive (non-sprouting) angiogenesis was first described in 1986 by Caduff et al.(Caduff JH et al. Scanning electron microscope study of the developing microvasculature in the postnatal rat lung. Anat Rec. 1986;216:154 64.) Intussusceptive angiogenesis is a highly dynamic process of microvascular growth observed within minutes to hours after a stimulus. The formation of a transluminal pillar is crucial for quick capillary plexus development.
Intussusceptive angiogenesis
Intussusceptive angiogenesis was observed during the formation and remodeling of vascular beds in many organs, including the mammary gland, the bone, the lungs, the glomeruli, the skeletal muscle, the ovaries, and others. This vascular expansion is particularly beneficial in tissues with high metabolic demand, ischemia, or hypoxia, as it enables the rapid delivery of nutrients and oxygen and the rapid removal of metabolic products. However, intussusceptive angiogenesis was observed not just as the physiological mechanism, but also in non-neoplastic (atherosclerosis, inflammatory diseases, malignancies, and viral infections) and neoplastic diseases. It, for example, plays a pivotal role in fibrotic remodeling in interstitial lung disease.
Ackermann et al. were the first to describe intussusceptive angiogenesis in the pathogenesis of COVID-19. They examined the lungs of seven people who died from COVID-19 and found a significant distortion of the lung angioarchitecture with prominent variations in the caliber of small vessels. Surprisingly, the lung capillaries showed cylindrical microstructures in the capillary lumina. In addition, they registered that the level of intussusceptive angiogenesis was increasing with the increasing duration of hospitalization. (Ackermann M, et al. Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in COVID-19. N Engl J Med. 2020; 383: 120–128. https://www.nejm.org/doi/full/10.1056/NEJMoa2015432 )
Stromal cell-derived factor-1 (SDF-1)/CXC motif chemokine receptor 4 (CXCR4) signaling is involved in blood vessel formation and remodeling by intussusception. Growing evidence implicates a pivotal role of the SDF-1/CXCR4 axis in myocardial repair, especially in myoangiogenesis after acute myocardial infarction and cardiomyopathy. Also, vascular-endothelial growth factor (VEGF) family members and their pathways play a critical role in both sprouting and intussusceptive angiogenesis. Previous studies showed that overexpression of VEGF and angiopoietins-1/2 increased the number of small holes in primordial capillary plexuses, a sign of increased intussusceptive angiogenesis. Also, VEGF concentration gradient disruption promotes shifting from sprouting to intussusceptive angiogenesis. These findings suggest an important role of VEGF in intussusceptive angiogenesis associated with COVID-19. (Madureira G & Soares R. The misunderstood link between SARS-CoV-2 and angiogenesis. A narrative review. Pulmonology, 2023, 29:4, 323-331. https://doi.org/10.1016/j.pulmoe.2021.08.004).Â
About the study
The researchers used conventional histopathology, immunohistochemistry, microvascular corrosion casting, scanning electron microscopy, phase-contrast synchrotron radiation tomographic microscopy, and gene expression analysis to assess morphological and molecular changes in the heart samples of patients who died of SARS-CoV-2 infection, pneumonia caused by influenza A virus subtype H1N1, and non-influenza/non-SARS-CoV-2 myocarditis (e.g., coxsackievirus). The study also included the heart samples of non-infected control cases. SARS-CoV-2 spike and nucleocapsid protein were detected by immunohistochemistry, and the presence of RNA was detected using an RNA-FISH probe and RT-PCR.
Myocarditis was defined according to the established Dallas criteria.
Results
Cardiac specimens were obtained from 24 COVID-19 cases (nine women and fifteen men), 16 influenza A subtype H1N1 patients (seven women and nine men), 8 non-influenza/non-SARS-CoV-2 myocarditis patients (three women and five men), and 9 control individuals (seven women and two men). COVID-19 patients were significantly older (73.7 ± 10.8 years) compared to influenza cases (52.3 ± 15.3 years), common myocarditis cases (54.8 ± 21.3), and non-inflamed control patients (54.9 ± 18.0).
Echocardiography data and serum levels of CK-MB, troponin, and NT-proBNP were available for 16/24 COVID-19 cases, 12/16 influenza cases, and 3/8 non-influenza myocarditis cases, respectively. Elevated serum markers, e.g., CK-MB, troponin, NT-proBNP, and/or abnormalities in echocardiography, e.g., reduced LVEF/RVEF or dyskinesia defined cardiac involvement.
From all available heart specimens, 13 from COVID-19 cases, 3 from non-influenza myocarditis cases, and 3 from control individuals were suitable for vascular corrosion casting and 3D scanning electron microscopy, the gold standard for detailed analysis of the microvasculature, angiogenesis, and vascular occlusions.
Light microscopy findings and cell infiltrates
Based on histopathologic findings, none of the hearts of COVID-19 patients met the established diagnostic criteria for viral myocarditis. 17/24 analyzed heart samples of COVID-19 patients were positive for SARS-CoV-2 RNA.
Analysis of inflammatory cell composition demonstrated a marked increase of macrophages (CD68 +) in cardiac specimens of all infectious groups compared to controls. The number of macrophages was twofold higher in the hearts from influenza cases than in those from COVID-19 patients and common myocarditis cases. In 18/24 COVID-19 hearts, the perivascular connective tissue displayed a diffuse infiltration with CD11b +  macrophages. In addition, cardiac specimens from COVID-19 cases had higher levels of TIE2-expressing macrophages (TieMs) than hearts from influenza and control cases. In 10/24 COVID-19 hearts, TIE2 was upregulated up to tenfold.
Lymphocytic infiltrates in COVID-19 and influenza cases were scarce. In contrast, all hearts of non-influenza/non-SARS-CoV-2 myocarditis cases showed lymphocytic infiltrates (severe inflammatory infiltrate in 3/8 cases, minor inflammatory infiltrate in 4/8 cases, and minor inflammatory infiltrate with single-cell necrosis in 1/8 cases). These marked lymphocytic inflammatory infiltrates were mixed (CD4, CD8, CD20).
Necrosis was not detected in the hearts of COVID-19, influenza, and control cases. Cardiomyocyte hypertrophy was most pronounced in the hearts of COVID-19 patients, followed by the controls, the influenza cases, and the cases with non-influenza/non-SARS-CoV-2 myocarditis. The level of interstitial fibrosis did not differ between the four groups.Â
Vascular remodeling
Conventional light microscopy and immunohistochemical staining for fibrin showed only scarce small vessel thrombi and no large vessel thrombi in the cardiac tissue of COVID-19 patients. Two hearts from the influenza group showed large vessel thrombi and no small vessel thrombi. In contrast to the results of light microscopy, SEM imaging of corrosion casts revealed a significantly higher quantity of ultrastructurally detectable thrombi in COVID-19 hearts, as indicated by abrupt breakoff of capillaries with a diameter of 1–3 µm, which are too small to be detected by conventional light microscopy.
A changed vascular architecture, a loss of vascular hierarchy, a tortuous arrangement, irregular sinusoidal vessel networks, and frequent vessel diameter alterations were all seen in heart samples from COVID-19 cases. Additionally, transluminal intussusceptive pillars were observed in the hearts of individuals who died of COVID-19, as evidenced by small holes in vascular corrosion casts at numerous vessel branches. Importantly, the presence of microthrombi positively correlated with the occurrence of intussusceptive pillars in COVID-19 hearts. This was not found in non-influenza myocarditis cases. Intussusceptive pillars, disturbed, feathered courses of heart fibers, and mild interstitial fibrosis were also identified using phase-contrast synchrotron radiation tomographic microscopy.
Vascular changes in COVID-19 hearts were observed at the level of the afferent, large-caliber arteries, in contrast to the regular vascular architecture seen in the hearts of control patients.
Gene expression analysis
In COVID-19 hearts, gene expression analysis identified significant upregulation of pro-inflammatory genes (IL1B, IL-6, IL8, and the toll-like receptor TLR2), hypoxia- and angiogenesis-related genes (VEGFC, FT1, and NOS3), and genes associated with monocyte recruitment (CXCR4, SDF-1, MMP9, CCR2, CXCR2, and MYD88). According to the authors, a marked increase in tissue macrophages and the upregulation of CCR2, CYCR2, CXCR4, and MYD88 in the hearts of COVID-19 cases suggest activation of local macrophages and upregulation of transcriptional profiles linked to the recruitment of circulating monocytes.
According to the analysis of 797 genes, there was a clear difference between heart samples from COVID-19 patients and those from influenza cases, but not between heart samples from COVID-19 patients and those from non-influenza/non-SARS-CoV-2 myocarditis cases. Heart samples of COVID-19 patients had twice as many differentially expressed genes as heart samples from influenza cases (152 genes in COVID-19 versus 72 genes in influenza). According to comparative gene analysis, genes for angiogenesis, cell migration, and epithelial-mesenchymal-transition (EMT) pathways, as signs of endothelial activation, were upregulated in COVID-19 hearts. The expression of genes for pro-inflammatory signaling and the antiviral response was the main characteristic of hearts from influenza cases.
Conclusion
This study demonstrated that cardiac involvement in COVID-19 manifested as perivascular infiltration with a subpopulation of CD11b+/TIE2+ macrophages, contributing to cardiac neoangiogenesis, rather than as conventional viral myocarditis defined by mononuclear infiltrates and myocyte damage. The most remarkable finding in the cardiac remodeling triggered by the SARS-CoV-2 infection was a marked neovascularization in the form of intussusceptive angiogenesis, which was associated with a significant infiltration of CD11b+/TIE2+ macrophages, which are drivers of intussusceptive angiogenesis. These findings support the view that macrophages are the main responders, from the recruitment of circulating monocytes to irreversible tissue remodeling in intussusceptive angiogenesis.
Researchers concluded that cardiac involvement in COVID-19 is an angiocentric macrophage-driven inflammatory process, distinct from classical anti-viral inflammatory response, and underestimated by conventional histopathologic analysis. Because of the irreversible alterations in the cardiac vasculature, systematic follow-up studies of long-term COVID cases are strongly recommended to identify potential post-acute cardiac symptoms and fibrotic remodeling.
Journal Reference
Werlein C, Ackermann M, Stark H et al. Inflammation and vascular remodeling in COVID-19 hearts. Angiogenesis (2023) 26:233–248. (Open Access)  https://doi.org/10.1007/s10456-022-09860-7
