Evidence for elastolytic activity in COVID-19 patients.
Recent studies linked alveolar epithelial senescence and impaired regeneration as inducing factors of fibrosis in COVID-19.17 However, the changes in the ECM that trigges in fibrosis in unknown. These studies reveal widespread alveolar-elastolysis in COVID-1 patients. Morphometrically, the elastin fibers in the normal alveoli appear as slender fibers aligned with the alveolar walls. The fibers often appear as bundles of similar length and branch at intersections between alveoli in non-COVID-19 lungs(Figure 2A) . Abundant elastin fibers also were observed at the tips of the alveolar septa. The elastin fibers that align with alveolar walls are essential in providing mechanical support and elastic recoil to the air spaces during respiration. The elastin fibers in COVID-19 lungs exhibited extensive disintegration (Figure 2B) . The degradation of elastin was most notable along the walls of alveoli that displayed extensive necrosis of alveolar epithelium. Further, fragmented elastin formed shorter, and single fibers that separated from the elastin bundles in the alveolar interstitial lining. The individual elastin fibers often lost their alignment within the alveolar walls. Quantitative measurement of the lengths of the elastin fibers displayed significant reduction in the lengths in the COVID-19 patients(Figure 2C) . In parallel, elastin fibers in the perivascular regions also displayed extensive cleavage in COVID-19 patients(Figure 2D). The loss of elastin along the blood vessels is associated with severe endothelial necrosis and distortion of the blood vessel structures. Thus, a loss of breathing capacity was compounded by the lack of elasticity in blood vessels carrying blood needed for gas exchange. In support of these findings, Western blot analysis of elastin from protein extracted from FFPE lung tissues displayed degradation of tropoelastin band at 70 kDa. Lung extracts from COVID-19 negative patients showed a prominent tropoelastin (Figure 2E) . Densitometry analysis of Western blots using ImageJ software(Figure 2F) showed a significant reduction in tropoelastin reactivity in COVID-19 lungs, in agreement with the morphometric analysis of elastin degradation in the COVID-19 lungs.
Collagen accumulates and replaces elastin in the lung parenchyma of COVID-19 patients.
Unlike the collagen staining that appeared mainly in the alveolar walls of non-COVID-19 patients, dense accumulations of collagen were observed in the alveolar interstitium in COVID-19 patients, accompanied by thickening of the alveolar septa (Figure 3A, B) . A quantitative assessment of collagen was determined using ImageJ analysis by selecting Trichrome-specific signals that show significant increase in collagen accumulation within the alveolar regions in the COVID-19 patient(Figure 3C) . In normal ECM of alveolar intersitium, elastin fibers are generally interspersed with collagen fibers that provides elasticity and mechanical strength to the alveoli. To evaluate the relation between collagen and elastin in COVID-19 patients, especially during elastolysis, this study examined expression of collagen and elastin fibers in the thin-alveolar walls of COVID-19 positive and COVID-19 negative patients. As shown in Figure 3 D-E , epithelial injury and elastin degradation in the alveolar walls were found replaced with abundant collagen deposition. The thickened walls of the alveoli show dense interstitial collagenous fibrosis associated with degradation or complete loss of elastin fibers (Figure 3 F-H) . Interestingly, the interstitial regions showing degraded elastin also display epithelial necrosis, indicating that both elastolysis and epithelial injury are preceding events in pathologic development of interstitial fibrosis (Figure 3 D-E) . A recent study identified collapse induration associated with alveolar epithelial necrosis and denudation of basal lamina in a COVID-19 patient.46Accordingly, this study found widespread alveolar collapse with hallmarks of necrotic epithelium and narrowing of the alveolar lumen. Interestingly, the collapsed alveoli also displayed an extensive degradation of elastin fibers and, conversely, dense collagen deposition in the collapsed alveolar regions (Figure 3I-J) suggesting that epithelial damage and elastolysis may precede the fibrotic changes in the injured alveoli. Similarly, perivascular regions showing loosely proliferating fibroblasts exhibit thick collagen deposition and degradation of elastin fibers (Figure S1H-J) . In support of these observations, the collagen:elastin ratio was increased by four-fold in the lung sections of COVID-19 patients compared to COVID-19 negative patients (Figure 4 A-E) . The lung parenchymal regions with advanced fibrosis displayed dense collagen diffusion with complete absence of elastin fibers, thus indicating an extensive elastolysis occurring during progression of the fibrosis in COVID-19 patients. Areas of fibrosing organizing pneumonia displayed extensive loss of alveolar epithelium that correlates with the loss of elastin fibers in the lung parenchyma (Figure 4 F-H) .