The fatal trajectory of pulmonary COVID-19 is driven by lobular ischemia and fibrotic remodelling.

BACKGROUND: COVID-19 is characterized by a heterogeneous clinical presentation, ranging from mild symptoms to severe courses of disease. 9-20% of hospitalized patients with severe lung disease die from COVID-19 and a substantial number of survivors develop long-COVID. Our objective was to provide comprehensive insights into the pathophysiology of severe COVID-19 and to identify liquid biomarkers for disease severity and therapy response. METHODS: We studied a total of 85 lungs (n = 31 COVID autopsy samples; n = 7 influenza A autopsy samples; n = 18 interstitial lung disease explants; n = 24 healthy controls) using the highest resolution Synchrotron radiation-based hierarchical phase-contrast tomography, scanning electron microscopy of microvascular corrosion casts, immunohistochemistry, matrix-assisted laser desorption ionization mass spectrometry imaging, and analysis of mRNA expression and biological pathways. Plasma samples from all disease groups were used for liquid biomarker determination using ELISA. The anatomic/molecular data were analyzed as a function of patients' hospitalization time. FINDINGS: The observed patchy/mosaic appearance of COVID-19 in conventional lung imaging resulted from microvascular occlusion and secondary lobular ischemia. The length of hospitalization was associated with increased intussusceptive angiogenesis. This was associated with enhanced angiogenic, and fibrotic gene expression demonstrated by molecular profiling and metabolomic analysis. Increased plasma fibrosis markers correlated with their pulmonary tissue transcript levels and predicted disease severity. Plasma analysis confirmed distinct fibrosis biomarkers (TSP2, GDF15, IGFBP7, Pro-C3) that predicted the fatal trajectory in COVID-19. INTERPRETATION: Pulmonary severe COVID-19 is a consequence of secondary lobular microischemia and fibrotic remodelling, resulting in a distinctive form of fibrotic interstitial lung disease that contributes to long-COVID. FUNDING: This project was made possible by a number of funders. The full list can be found within the Declaration of interests / Acknowledgements section at the end of the manuscript.

Exploring m6A-RNA methylation as a potential therapeutic strategy for acute lung injury and acute respiratory distress syndrome.

N6-methyladenosine (m6A) is the most common methylation modification in mammalian messenger RNA (mRNA) and noncoding RNAs. m6A modification plays a role in the regulation of gene expression and deregulation of m6A methylation has been implicated in many human diseases. Recent publications suggest that exploitation of this methylation process may possess utility against acute lung injury (ALI). ALI and its more severe form, acute respiratory distress syndrome (ARDS) are acute, inflammatory clinical syndromes characterized by poor oxygenation and diffuse pulmonary infiltrates. This syndrome is associated with microvascular endothelial dysfunction, subsequent pulmonary hypertension and may ultimately lead to mortality without rigorous and acute clinical intervention. Over the years, many attempts have been made to detect novel therapeutic avenues for research without much success. The urgency for the discovery of novel therapeutic agents has become more pronounced recently given the current pandemic infection of coronavirus disease 2019 (COVID-2019), still ongoing at the time that this review is being written. We review the current landscape of literature regarding ALI and ARDS etiology, pathophysiology, and therapeutics and present a potential role of m6A methylation. Additionally, we will establish the axiomatic principles of m6A methylation to provide a framework. In conclusion, METTL3, or methyltransferase-like 3, the selective RNA methyltransferase for m6A, is a hub of proinflammatory gene expression regulation in ALI, and using a modern drug discovery strategy will identify new and effective ALI drug candidates targeting METTTL3.

Inflammation and vascular remodeling in COVID-19 hearts.

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.

SARS-CoV-2 spike protein induces IL-18-mediated cardiopulmonary inflammation via reduced mitophagy.

Cardiopulmonary complications are major drivers of mortality caused by the SARS-CoV-2 virus. Interleukin-18, an inflammasome-induced cytokine, has emerged as a novel mediator of cardiopulmonary pathologies but its regulation via SARS-CoV-2 signaling remains unknown. Based on a screening panel, IL-18 was identified amongst 19 cytokines to stratify mortality and hospitalization burden in patients hospitalized with COVID-19. Supporting clinical data, administration of SARS-CoV-2 Spike 1 (S1) glycoprotein or receptor-binding domain (RBD) proteins into human angiotensin-converting enzyme 2 (hACE2) transgenic mice induced cardiac fibrosis and dysfunction associated with higher NF-κB phosphorylation (pNF-κB) and cardiopulmonary-derived IL-18 and NLRP3 expression. IL-18 inhibition via IL-18BP resulted in decreased cardiac pNF-κB and improved cardiac fibrosis and dysfunction in S1- or RBD-exposed hACE2 mice. Through in vivo and in vitro work, both S1 and RBD proteins induced NLRP3 inflammasome and IL-18 expression by inhibiting mitophagy and increasing mitochondrial reactive oxygenation species. Enhancing mitophagy prevented Spike protein-mediated IL-18 expression. Moreover, IL-18 inhibition reduced Spike protein-mediated pNF-κB and EC permeability. Overall, the link between reduced mitophagy and inflammasome activation represents a novel mechanism during COVID-19 pathogenesis and suggests IL-18 and mitophagy as potential therapeutic targets.

RAC1 nitration at Y32 IS involved in the endothelial barrier disruption associated with lipopolysaccharide-mediated acute lung injury.

Acute lung injury (ALI), a devastating illness induced by systemic inflammation e.g., sepsis or local lung inflammation e.g., COVID-19 mediated severe pneumonia, has an unacceptably high mortality and has no effective therapy. ALI is associated with increased pulmonary microvascular hyperpermeability and alveolar flooding. The small Rho GTPases, RhoA and Rac1 are central regulators of vascular permeability through cytoskeleton rearrangements. RhoA and Rac1 have opposing functional outcome: RhoA induces an endothelial contractile phenotype and barrier disruption, while Rac1 stabilizes endothelial junctions and increases barrier integrity. In ALI, RhoA activity is increased while Rac1 activity is reduced. We have shown that the activation of RhoA in lipopolysaccharide (LPS)-mediated ALI, is dependent, at least in part, on a single nitration event at tyrosine (Y)34. Thus, the purpose of this study was to determine if the inhibition of Rac1 is also dependent on its nitration. Our data show that Rac1 inhibition by LPS is associated with its nitration that mass spectrometry identified as Y32, within the switch I region adjacent to the nucleotide-binding site. Using a molecular modeling approach, we designed a nitration shielding peptide for Rac1, designated NipR2 (nitration inhibitor peptide for the Rho GTPases 2), which attenuated the LPS-induced nitration of Rac1 at Y32, preserves Rac1 activity and attenuates the LPS-mediated disruption of the endothelial barrier in human lung microvascular endothelial cells (HLMVEC). Using a murine model of ALI induced by intratracheal installation of LPS we found that NipR2 successfully prevented Rac1 nitration and Rac1 inhibition, and more importantly attenuated pulmonary inflammation, reduced lung injury and prevented the loss of lung function. Together, our data identify a new post-translational mechanism of Rac1 inhibition through its nitration at Y32. As NipR2 also reduces sepsis induced ALI in the mouse lung, we conclude that Rac1 nitration is a therapeutic target in ALI.