Buckling forces and the wavy folds between pleural epithelial cells.

Cell shapes in tissues are affected by the biophysical interaction between cells. Tissue forces can influence specific cell features such as cell geometry and cell surface area. Here, we examined the 2-dimensional shape, size, and perimeter of pleural epithelial cells at various lung volumes. We demonstrated a 1.53-fold increase in 2-dimensional cell surface area and a 1.43-fold increase in cell perimeter at total lung capacity compared to residual lung volume. Consistent with previous results, close inspection of the pleura demonstrated wavy folds between pleural epithelial cells at all lung volumes. To investigate a potential explanation for the wavy folds, we developed a physical simulacrum suggested by D'Arcy Thompson in On Growth and Form. The simulacrum suggested that the wavy folds were the result of redundant cell membranes unable to contract. To test this hypothesis, we developed a numerical simulation to evaluate the impact of an increase in 2-dimensional cell surface area and cell perimeter on the shape of the cell-cell interface. Our simulation demonstrated that an increase in cell perimeter, rather than an increase in 2-dimensional cell surface area, had the most direct impact on the presence of wavy folds. We conclude that wavy folds between pleural epithelial cells reflects buckling forces arising from the excess cell perimeter necessary to accommodate visceral organ expansion.

The role of vasculature and angiogenesis in respiratory diseases.

In European countries, nearly 10% of all hospital admissions are related to respiratory diseases, mainly chronic life-threatening diseases such as COPD, pulmonary hypertension, IPF or lung cancer. The contribution of blood vessels and angiogenesis to lung regeneration, remodeling and disease progression has been increasingly appreciated. The vascular supply of the lung shows the peculiarity of dual perfusion of the pulmonary circulation (vasa publica), which maintains a functional blood-gas barrier, and the bronchial circulation (vasa privata), which reveals a profiled capacity for angiogenesis (namely intussusceptive and sprouting angiogenesis) and alveolar-vascular remodeling by the recruitment of endothelial precursor cells. The aim of this review is to outline the importance of vascular remodeling and angiogenesis in a variety of non-neoplastic and neoplastic acute and chronic respiratory diseases such as lung infection, COPD, lung fibrosis, pulmonary hypertension and lung cancer.

Evolving topological order in the postnatal visceral pleura.

BACKGROUND: Changes in epithelial cell shape reflects optimal cell packing and the minimization of surface free energy, but also cell-cell interactions, cell proliferation, and cytoskeletal rearrangements. RESULTS: Here, we studied the structure of the rat pleura in the first 15 days after birth. After pleural isolation and image segmentation, the analysis demonstrated a progression of epithelial order from postnatal day 1 (P1) to P15. The cells with the largest surface area and greatest shape variability were observed at P1. In contrast, the cells with the smallest surface area and most shape consistency were observed at P15. A comparison of polygonal cell geometries demonstrated progressive optimization with an increase in the number of hexagons (six-sided) as well as five-sided and seven-sided polygons. Analysis of the epithelial organization with Voronoi tessellations and graphlet motif frequencies demonstrated a developmental path strikingly distinct from mathematical and natural reference paths. Graph Theory analysis of cell connectivity demonstrated a progressive decrease in network heterogeneity and clustering coefficient from P1 to P15. CONCLUSIONS: We conclude that the rat pleura undergoes a striking change in pleural structure from P1 to P15. Further, a geometric and network-based approach can provide a quantitative characterization of these developmental changes.

Vascularization of the adult mouse lung grafted onto the chick chorioallantoic membrane.

In the later stages of angiogenesis, the vascular sprout transitions into a functional vessel by fusing with a target vessel. Although this process appears to routinely occur in embryonic tissue, the biologic rules for sprout fusion and lumenization in adult regenerating tissue are unknown. To investigate this process, we grafted portions of the regenerating post-pneumonectomy lung onto the chick chorioallantoic membrane (CAM). Grafts from all 4 lobes of the post-pneumonectomy right lung demonstrated peri-graft angiogenesis as reflected by fluorescent plasma markers; however, fluorescent microsphere perfusion primarily occurred in the lobe of the lung that is the dominant site of post-pneumonectomy angiogenesis-namely, the cardiac lobe. Vascularization of the cardiac lobe grafts was confirmed by active tissue growth (p < .05). Functional vascular connections between the cardiac lobe and the CAM vascular network were demonstrated by confocal fluorescence microscopy as well as corrosion casting and scanning electron microscopy (SEM). Bulk transcriptional profiling of the cardiac lobe demonstrated the enhanced expression of many genes relative to alveolar epithelial cell (CD11b-/CD31-) control cells, but only the upregulation of Ereg and Fgf6 compared to the less well-vascularized right upper lobe. The growth of actively regenerating non-neoplastic adult tissue on the CAM demonstrates that functional lumenization can occur between species (mouse and chick) and across the developmental spectrum (adult and embryo).

Stochastic simulations of self-organized elastogenesis in the developing lung.

In the normal lung, the dominant cable is an elastic "line element" composed of elastin fibers bound to a protein scaffold. The cable line element maintains alveolar geometry by balancing surface forces within the alveolus and changes in lung volume with exercise. Recent work in the postnatal rat lung has suggested that the process of cable development is self-organized in the extracellular matrix. Early in postnatal development, a blanket of tropoelastin (TE) spheres appear in the primitive lung. Within 7 to 10 days, the TE spheres are incorporated into a distributed protein scaffold creating the mature cable line element. To study the process of extracellular assembly, we used cellular automata (CA) simulations. CA simulations demonstrated that the intermediate step of tropoelastin self-aggregation into TE spheres enhanced the efficiency of cable formation more than 5-fold. Similarly, the rate of tropoelastin production had a direct impact on the efficiency of scaffold binding. The binding affinity of the tropoelastin to the protein scaffold, potentially reflecting heritable traits, also had a significant impact on cable development. In contrast, the spatial distribution of TE monomer production, increased Brownian motion and variations in scaffold geometry did not significantly impact simulations of cable development. We conclude that CA simulations are useful in exploring the impact of concentration, geometry, and movement on the fundamental process of elastogenesis.

Geometric and network organization of visceral organ epithelium.

Mammalian epithelia form a continuous sheet of cells that line the surface of visceral organs. To analyze the epithelial organization of the heart, lung, liver and bowel, epithelial cells were labeled in situ, isolated as a single layer and imaged as large epithelial digitally combine montages. The stitched epithelial images were analyzed for geometric and network organization. Geometric analysis demonstrated a similar polygon distribution in all organs with the greatest variability in the heart epithelia. Notably, the normal liver and inflated lung demonstrated the largest average cell surface area (p < 0.01). In lung epithelia, characteristic wavy or interdigitated cell boundaries were observed. The prevalence of interdigitations increased with lung inflation. To complement the geometric analyses, the epithelia were converted into a network of cell-to-cell contacts. Using the open-source software EpiGraph, subgraph (graphlet) frequencies were used to characterize epithelial organization and compare to mathematical (Epi-Hexagon), random (Epi-Random) and natural (Epi-Voronoi5) patterns. As expected, the patterns of the lung epithelia were independent of lung volume. In contrast, liver epithelia demonstrated a pattern distinct from lung, heart and bowel epithelia (p < 0.05). We conclude that geometric and network analyses can be useful tools in characterizing fundamental differences in mammalian tissue topology and epithelial organization.

Biomechanics of a Plant-Derived Sealant for Corneal Injuries.

Purpose: The corneal epithelium has a glycocalyx composed of membrane-associated glycoproteins, mucins, and galactin-3. Similar to the glycocalyx in visceral tissues, the corneal glycocalyx functions to limit fluid loss and minimize frictional forces. Recently, the plant-derived heteropolysaccharide pectin has been shown to physically entangle with the visceral organ glycocalyx. The ability of pectin to entangle with the corneal epithelium is unknown. Methods: To explore the potential role of pectin as a corneal bioadhesive, we assessed the adhesive characteristics of pectin films in a bovine globe model. Results: Pectin film was flexible, translucent, and low profile (80 µm thick). Molded in tape form, pectin films were significantly more adherent to the bovine cornea than control biopolymers of nanocellulose fibers, sodium hyaluronate, and carboxymethyl cellulose (P < 0.05). Adhesion strength was near maximal within seconds of contact. Compatible with wound closure under tension, the relative adhesion strength was greatest at a peel angle less than 45 degrees. The corneal incisions sealed with pectin film were resistant to anterior chamber pressure fluctuations ranging from negative 51.3 ± 8.9 mm Hg to positive 214 ± 68.6 mm Hg. Consistent with these findings, scanning electron microscopy demonstrated a low-profile film densely adherent to the bovine cornea. Finally, the adhesion of the pectin films facilitated the en face harvest of the corneal epithelium without physical dissection or enzymatic digestion. Conclusions: We conclude that pectin films strongly adhere to the corneal glycocalyx. Translational Relevance: The plant-derived pectin biopolymer provides potential utility for corneal wound healing as well as targeted drug delivery.

3D stimulated Raman spectral imaging of water dynamics associated with pectin-glycocalyceal entanglement.

Pectin is a heteropolysaccharide responsible for the structural integrity of the cell walls of terrestrial plants. When applied to the surface of mammalian visceral organs, pectin films form a strong physical bond with the surface glycocalyx. A potential mechanism of pectin adhesion to the glycocalyx is the water-dependent entanglement of pectin polysaccharide chains with the glycocalyx. A better understanding of such fundamental mechanisms regarding the water transport dynamics in pectin hydrogels is of importance for medical applications, e.g., surgical wound sealing. We report on the water transport dynamics in hydrating glass-phase pectin films with particular emphasis on the water content at the pectin-glycocalyceal interface. We used label-free 3D stimulated Raman scattering (SRS) spectral imaging to provide insights into the pectin-tissue adhesive interface without the confounding effects of sample fixation, dehydration, shrinkage, or staining.

Topography of pleural epithelial structure enabled by en face isolation and machine learning.

Pleural epithelial adaptations to mechanical stress are relevant to both normal lung function and parenchymal lung diseases. Assessing regional differences in mechanical stress, however, has been complicated by the nonlinear stress-strain properties of the lung and the large displacements with ventilation. Moreover, there is no reliable method of isolating pleural epithelium for structural studies. To define the topographic variation in pleural structure, we developed a method of en face harvest of murine pleural epithelium. Silver-stain was used to highlight cell borders and facilitate imaging with light microscopy. Machine learning and watershed segmentation were used to define the cell area and cell perimeter of the isolated pleural epithelial cells. In the deflated lung at residual volume, the pleural epithelial cells were significantly larger in the apex (624 ± 247 µm2 ) than in basilar regions of the lung (471 ± 119 µm2 ) (p < 0.001). The distortion of apical epithelial cells was consistent with a vertical gradient of pleural pressures. To assess epithelial changes with inflation, the pleura was studied at total lung capacity. The average epithelial cell area increased 57% and the average perimeter increased 27% between residual volume and total lung capacity. The increase in lung volume was less than half the percent change predicted by uniform or isotropic expansion of the lung. We conclude that the structured analysis of pleural epithelial cells complements studies of pulmonary microstructure and provides useful insights into the regional distribution of mechanical stresses in the lung.

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.

Facilitated Transport across Glycocalyceal Barriers in the Chick Chorioallantoic Membrane.

Targeted drug delivery to visceral organs offers the possibility of not only limiting the required dose, but also minimizing drug toxicity; however, there is no reliable method for delivering drugs to the surface of visceral organs. Here, we used six color tracers and the chick chorioallantoic membrane (CAM) model to investigate the use of the heteropolysaccharide pectin to facilitate tracer diffusion across the glycocalyceal charge barrier. The color tracers included brilliant blue, Congo red, crystal violet, indocyanine green, methylene blue, and methyl green. The direct application of the tracers to the CAM surface or embedding tracers into linear-chain nanocellulose fiber films resulted in no significant diffusion into the CAM. In contrast, when the tracers were actively loaded into branched-chain pectin films, there was significant detectable diffusion of the tracers into the CAM. The facilitated diffusion was observed in the three cationic tracers but was limited in the three anionic tracers. Diffusion appeared to be dependent on ionic charge, but independent of tracer size or molecular mass. We conclude that dye-loaded pectin films facilitated the diffusion of color tracers across the glycocalyceal charge barrier and may provide a therapeutic path for drug delivery to the surface of visceral organs.

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.

Kinetics of Pectin Biopolymer Facial Erosion Characterized by Fluorescent Tracer Microfluidics.

Pectin is a plant-derived heteropolysaccharide that has been implicated in drug development, tissue engineering, and visceral organ repair. Pectin demonstrates remarkable biostability in a variety of physiologic environments but is biodegradable in water. To understand the dynamics of pectin biodegradation in basic environments, we developed a microfluidics system that facilitated the quantitative comparison of pectin films exposed to facial erosion. Pectin biodegradation was assessed using fluorescein tracer embedded in pectin, trypan blue quenching of released fluorescence, and highly sensitive microfluorimetry. The microfluidic perfusate, delivered through 6 um-pore synthetic membrane interface, demonstrated nonlinear erosion of the pectin film; 75% of tracer was released in 28 h. The microfluidics system was used to identify potential modifiers of pectin erosion. The polyphenolic compound tannic acid, loaded into citrus pectin films, demonstrated a dose-dependent decrease in pectin erosion. Tannic acid had no detectable impact on the physical properties of citrus pectin including adhesivity and cohesion. In contrast, tannic acid weakened the burst strength and cohesion of pectins derived from soy bean and potato sources. We conclude that facial erosion may explain the biostability of citrus pectin on visceral organ surfaces as well as provide a useful method for identifying modifiers of citrus pectin biodegradation.

Endothelialitis, Microischemia, and Intussusceptive Angiogenesis in COVID-19.

COVID-19 has been associated with a range of illness severity-from minimal symptoms to life-threatening multisystem organ failure. The severe forms of COVID-19 appear to be associated with an angiocentric or vascular phase of the disease. In studying autopsy patients succumbing to COVID-19, we found alveolar capillary microthrombi were 9 times more common in COVID-19 than in comparable patients with influenza. Corrosion casting of the COVID-19 microcirculation has revealed microvascular distortion, enhanced bronchial circulation, and striking increases in intussusceptive angiogenesis. In patients with severe COVID-19, endothelial cells commonly demonstrate significant ultrastructural injury. High-resolution imaging suggests that microcirculation perturbations are linked to ischemic changes in microanatomic compartments of the lung (secondary lobules). NanoString profiling of these regions has confirmed a transcriptional signature compatible with microischemia. We conclude that irreversible tissue ischemia provides an explanation for the cystic and fibrotic changes associated with long-haul COVID-19 symptoms.

Optical and Mechanical Properties of Self-Repairing Pectin Biopolymers.

Pectin's unique physicochemical properties have been linked to a variety of reparative and regenerative processes in nature. To investigate the effect of water on pectin repair, we used a 5 mm stainless-steel uniaxial load to fracture glass phase pectin films. The fractured gel phase films were placed on a 1.5-1.8 mm thick layer of water and incubated for 8 h at room temperature and ambient humidity. There was no immersion or agitation. The repaired pectin film was subsequently assessed for its optical and mechanical properties. Light microscopy demonstrated repair of the detectable fracture area and restoration of the films' optical properties. The burst strength of the repaired film declined to 55% of the original film. However, its resilience was restored to 87% of the original film. Finally, a comparison of the initial and post-repair fracture patterns demonstrated no recurrent fissures in the repaired glass phase films. The water-induced repair of the pectin film was superior to the optical and mechanical properties of the repaired films composed of nanocellulose fibers, sodium hyaluronate, and oxidized cellulose. We conclude that the unique physicochemical properties of pectin facilitate the water-induced self-repair of fractured pectin films.

Interstitial lung disease in infancy and early childhood: a clinicopathological primer.

Children's interstitial lung disease (chILD) encompasses a wide and heterogeneous spectrum of diseases substantially different from that of adults. Established classification systems divide chILD into conditions more prevalent in infancy and other conditions occurring at any age. This categorisation is based on a multidisciplinary approach including clinical, radiological, genetic and histological findings. The diagnostic evaluation may include lung biopsies if other diagnostic approaches failed to identify a precise chILD entity, or if severe or refractory respiratory distress of unknown cause is present. As the majority of children will be evaluated and diagnosed outside of specialist centres, this review summarises relevant clinical, genetic and histological findings of chILD to provide assistance in clinical assessment and rational diagnostics.

Active Loading of Pectin Hydrogels for Targeted Drug Delivery.

Hydrogels provide a promising method for the targeted delivery of protein drugs. Loading the protein drug into the hydrogel free volume can be challenging due to limited quantities of the drug (e.g., growth factor) and complex physicochemical properties of the hydrogel. Here, we investigated both passive and active loading of the heteropolysaccharide hydrogel pectin. Passive loading of glass phase pectin films was evaluated by contact angles and fractional thickness of the pectin films. Four pectin sources demonstrated mean contact angles of 88° with water and 122° with pleural fluid (p < 0.05). Slow kinetics and evaporative losses precluded passive loading. In contrast, active loading of the translucent pectin films was evaluated with the colorimetric tracer methylene blue. Active loading parameters were systematically varied and recorded at 500 points/s. The distribution of the tracer was evaluated by image morphometry. Active loading of the tracer into the pectin films required the optimization of probe velocity, compression force, and contact time. We conclude that active loading using pectin-specific conditions is required for the efficient embedding of low viscosity liquids into pectin hydrogels.

Noninvasive Method and Metric for Monitoring Lung Condition.

For hospitalized patients with pulmonary conditions, the onset of respiratory decline can occur unnoticed, due to the absence of a way to continuously and noninvasively monitor lung condition. Based on the relationship between lung volume and pleural pressure, we hypothesized that the time delay (∆t) between the start of a respiratory cycle and the occurrence of lung sounds associated with inspiration would correlate with lung volume. Additionally, we developed a re-search tool, consisting of a respiration belt, digital stethoscope, data collection system and MATLAB algorithm, to measure this delay. We conducted a feasibility study with three healthy individuals that involved safely manipulating lung volume, through subject position and activity, and plotting ∆t against volume measurements obtained via spirometry. The results indicated that ∆t was measurable and changed with lung volume and, therefore, has the potential to serve as a lung condition monitoring tool.