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) .