hLMSC Secretome Affects Macrophage Activity Differentially Depending on Lung-Mimetic Environments

Mesenchymal stromal cell (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate the activity of macrophages. Despite recent advances, there is scarce information regarding changes of the secretome content attributed to physiomimetic cultures and, especially, how secretome content influence on macrophage activity for therapy. hLMSCs from human donors were cultured on devices developed in house that enabled lung-mimetic strain. hLMSC secretome was analyzed for typical cytokines, chemokines and growth factors. RNA was analyzed for the gene expression of CTGF and CYR61. Human monocytes were differentiated to macrophages and assessed for their phagocytic capacity and for M1/M2 subtypes by the analysis of typical cell surface markers in the presence of hLMSC secretome. CTGF and CYR61 displayed a marked reduction when cultured in lung-derived hydrogels (L-Hydrogels). The secretome showed that lung-derived scaffolds had a distinct secretion while there was a large overlap between L-Hydrogel and the conventionally (2D) cultured samples. Additionally, secretome from L-Scaffold showed an HGF increase, while IL-6 and TNF-α decreased in lung-mimetic environments. Similarly, phagocytosis decreased in a lung-mimetic environment. L-Scaffold showed a decrease of M1 population while stretch upregulated M2b subpopulations. In summary, mechanical features of the lung ECM and stretch orchestrate anti-inflammatory and immunosuppressive outcomes of hLMSCs

Converging pathways in pulmonary fibrosis and Covid-19 - The fibrotic link to disease severity : Common molecular pathways in Covid-19 and pulmonary fibrosis

As Covid-19 affects millions of people worldwide, the global health care will encounter an increasing burden of the aftermaths of the disease. Evidence shows that up to a fifth of the patients develop fibrotic tissue in the lung. The SARS outbreak in the early 2000 resulted in chronic pulmonary fibrosis in a subset (around 4%) of the patients, and correlated to reduced lung function and forced expiratory volume (FEV). The similarities between corona virus infections causing SARS and Covid-19 are striking, except that the novel coronavirus, SARS-CoV-2, has proven to have an even higher communicability. This would translate into a large number of patients seeking care for clinical signs of pulmonary fibrosis, given that the Covid-19 pandemic has up till now (Sept 2020) affected around 30 million people. The SARS-CoV-2 is dependent on binding to the angiotensin converting enzyme 2 (ACE2), which is part of the renin-angiotensin system (RAS). Downregulation of ACE2 upon virus binding disturbs downstream activities of RAS resulting in increased inflammation and development of fibrosis. The poor prognosis and risk of developing pulmonary fibrosis are therefore associated with the increased expression of ACE2 in risk groups, such as obesity, heart disorders and aging, conferring plenty of binding opportunity for the virus and subsequently the internalization of ACE2, thus devoiding the enzyme from acting counter-inflammatory and antifibrotic. Identifying pathways that are associated with Covid-19 severity that result in pulmonary fibrosis may enable early diagnosis and individualized treatment for these patients to prevent or reduce irreversible fibrotic damage to the lung

Glycosaminoglycans: a link between development and regeneration in the lung : a link between development and regeneration in the lung

What can we learn from embryogenesis to increase our understanding of how regeneration of damaged adult lung tissue could be induced in serious lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and asthma? The local tissue niche determines events in both embryogenesis and repair of the adult lung. Important constituents of the niche are extracellular matrix (ECM) molecules including proteoglycans and glycosaminoglycans (GAG). GAGs, strategically located in the pericellular and extracellular space, bind developmentally active growth factors and morphogenes such as fibroblast growth factors (FGF), transforming growth factor- (TGF-) and bone morphogenetic proteins (BMPs) aside from cytokines. These interactions affect activities in many cells, including stem cells, important in development and tissue regeneration. Moreover, it is becoming clear that the "inherent code", such as sulfation of disaccharides of GAGs is a strong determinant of cellular outcome. Sulfation pattern, deacetylations and epimerizations of GAG chains function as tuning forks in gradient formation of morphogens, growth factors and cytokines. Learning to tune these fine instruments, i.e. interactions between growth factors (GF), chemokines and cytokines with the specific disaccharide code of GAGs in the adult lung, could become the key to unlock inherent regenerative forces to override pathological remodeling. This review aims to give an overview of the role GAGs play during development and similar events in regenerative efforts in the adult lung

The proportion of alveolar type 1 cells decreases in murine hypoplastic congenital diaphragmatic hernia lungs (vol 14, e0214793, 2019)

[This corrects the article DOI: 10.1371/journal.pone.0214793.].status: publishe

The proportion of alveolar type 1 cells decreases in murine hypoplastic congenital diaphragmatic hernia lungs

Background Pulmonary hypoplasia, characterized by incomplete alveolar development, remains a major cause of mortality and morbidity in congenital diaphragmatic hernia. Recently demonstrated to differentiate from a common bipotent progenitor during development, the two cell types that line the alveoli type 1 and type 2 alveolar cells have shown to alter their relative ratio in congenital diaphragmatic hernia lungs. Objective We used the nitrofen/bisdiamine mouse model to induce congenital diaphragmatic hernia and accurately assess the status of alveolar epithelial cell differentiation in relation to the common bipotent progenitors. Study design Pregnant Swiss mice were gavage-fed with nitrofen/bisdiamine or vehicle at embryonic day 8.5. The administered dose was optimized by assessing the survival, congenital diaphragmatic hernia and facial abnormality rates of the exposed mouse pups. NanoCT was performed on embryonic day 11.5 and 16.5 to assess the embryonic and early canalicular stages of lung development. At embryonic day 17.5 corresponding to late canalicular stage, congenital diaphragmatic hernia lungs were characterized by measuring the lung weight/ body weight ratio, morphometry, epithelial cell marker gene expression levels and alveolar cell type quantification. Results Nitrofen/bisdiamine associated congenital diaphragmatic hernia lungs showed delayed development, hypoplasia with morphologic immaturity and thickened alveolar walls. Expression levels of distal epithelial progenitor marker Id2 increased, alveolar type 1 cell markers Pdpn and Hopx decreased, while type 2 cell markers pro-SPC and Muc1 remained constant during the canalicular stage. The number of Pdpn + type 1 alveolar cells also decreased in congenital diaphragmatic hernia lungs. Conclusion The mouse nitrofen/bisdiamine model is a potential model allowing the study of congenital diaphragmatic hernia lung development from early stages using a wide array of methods. Based on this model, the alveolar epithelium showed a decrease in the number of alveolar type 1 cell in congenital diaphragmatic hernia lungs while type 2 cell population remains unchanged

Distal lung microenvironment triggers release of mediators recognized as potential systemic biomarkers for idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an unmet need of biomarkers that can aid in the diagnostic and prognostic assessment of the disease and response to treatment. In this two-part explorative proteomic study, we demonstrate how proteins associated with tissue remodeling, inflammation and chemotaxis such as MMP7, CXCL13 and CCL19 are released in response to aberrant extracellular matrix (ECM) in IPF lung. We used a novel ex vivo model where decellularized lung tissue from IPF patients and healthy donors were repopulated with healthy fibroblasts to monitor locally released mediators. Results were validated in longitudinally collected serum samples from 38 IPF patients and from 77 healthy controls. We demonstrate how proteins elevated in the ex vivo model (e.g., MMP7), and other serum proteins found elevated in IPF patients such as HGF, VEGFA, MCP-3, IL-6 and TNFRSF12A, are associated with disease severity and progression and their response to antifibrotic treatment. Our study supports the model’s applicability in studying mechanisms involved in IPF and provides additional evidence for both established and potentially new biomarkers in IPF

Harnessing the ECM Microenvironment to Ameliorate Mesenchymal Stromal Cell-Based Therapy in Chronic Lung Diseases

It is known that the cell environment such as biomechanical properties and extracellular matrix (ECM) composition dictate cell behaviour including migration, proliferation, and differentiation. Important constituents of the microenvironment, including ECM molecules such as proteoglycans and glycosaminoglycans (GAGs), determine events in both embryogenesis and repair of the adult lung. Mesenchymal stromal/stem cells (MSC) have been shown to have immunomodulatory properties and may be potent actors regulating tissue remodelling and regenerative cell responses upon lung injury. Using MSC in cell-based therapy holds promise for treatment of chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). However, so far clinical trials with MSCs in COPD have not had a significant impact on disease amelioration nor on IPF, where low cell survival rate and pulmonary retention time are major hurdles to overcome. Research shows that the microenvironment has a profound impact on transplanted MSCs. In our studies on acellular lung tissue slices (lung scaffolds) from IPF patients versus healthy individuals, we see a profound effect on cellular activity, where healthy cells cultured in diseased lung scaffolds adapt and produce proteins further promoting a diseased environment, whereas cells on healthy scaffolds sustain a healthy proteomic profile. Therefore, modulating the environmental context for cell-based therapy may be a potent way to improve treatment using MSCs. In this review, we will describe the importance of the microenvironment for cell-based therapy in chronic lung diseases, how MSC-ECM interactions can affect therapeutic output and describe current progress in the field of cell-based therapy

The proportion of alveolar type 1 cells decreases in murine hypoplastic congenital diaphragmatic hernia lungs

BACKGROUND: Pulmonary hypoplasia, characterized by incomplete alveolar development, remains a major cause of mortality and morbidity in congenital diaphragmatic hernia. Recently demonstrated to differentiate from a common bipotent progenitor during development, the two cell types that line the alveoli type 1 and type 2 alveolar cells have shown to alter their relative ratio in congenital diaphragmatic hernia lungs. OBJECTIVE: We used the nitrofen/bisdiamine mouse model to induce congenital diaphragmatic hernia and accurately assess the status of alveolar epithelial cell differentiation in relation to the common bipotent progenitors. STUDY DESIGN: Pregnant Swiss mice were gavage-fed with nitrofen/bisdiamine or vehicle at embryonic day 8.5. The administered dose was optimized by assessing the survival, congenital diaphragmatic hernia and facial abnormality rates of the exposed mouse pups. NanoCT was performed on embryonic day 11.5 and 16.5 to assess the embryonic and early canalicular stages of lung development. At embryonic day 17.5 corresponding to late canalicular stage, congenital diaphragmatic hernia lungs were characterized by measuring the lung weight/body weight ratio, morphometry, epithelial cell marker gene expression levels and alveolar cell type quantification. RESULTS: Nitrofen/bisdiamine associated congenital diaphragmatic hernia lungs showed delayed development, hypoplasia with morphologic immaturity and thickened alveolar walls. Expression levels of distal epithelial progenitor marker Id2 increased, alveolar type 1 cell markers Pdpn and Hopx decreased, while type 2 cell markers pro-SPC and Muc1 remained constant during the canalicular stage. The number of Pdpn+ type 1 alveolar cells also decreased in congenital diaphragmatic hernia lungs. CONCLUSION: The mouse nitrofen/bisdiamine model is a potential model allowing the study of congenital diaphragmatic hernia lung development from early stages using a wide array of methods. Based on this model, the alveolar epithelium showed a decrease in the number of alveolar type 1 cell in congenital diaphragmatic hernia lungs while type 2 cell population remains unchanged.status: publishe

Stretch increases alveolar type 1 cell number in fetal lungs through ROCK-Yap/Taz pathway

Accurate fluid pressure in the fetal lung is critical for its development, especially at the beginning of the saccular stage when alveolar epithelial type 1 (AT1) and type 2 (AT2) cells differentiate from the epithelial progenitors. Despite our growing understanding of the role of physical forces in lung development, the molecular mechanisms that regulate the transduction of mechanical stretch to alveolar differentiation remain elusive. To simulate lung distension, we optimized both an ex vivo model with precision cut lung slices and an in vivo model of fetal tracheal occlusion. Increased mechanical tension showed to improve alveolar maturation and differentiation toward AT1. By manipulating ROCK pathway, we demonstrate that stretch-induced Yap/Taz activation promotes alveolar differentiation toward AT1 phenotype via ROCK activity. Our findings show that balanced ROCK-Yap/Taz signaling is essential to regulate AT1 differentiation in response to mechanical stretching of the fetal lung, which might be helpful in improving lung development and regeneration