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Abstract Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor‐dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre‐analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood‐EV), a tool to record and report information about pre‐analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre‐analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence‐based optimization of pre‐analytical protocols through meta‐analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter‐laboratory comparative EV studies, and (iv) improve the peer review process.

Lung fibroblasts contribute to asthma pathology partly through modulation of the immune environment in the airway. Tumor necrosis factor-α (TNFα) expression is upregulated in asthmatic lungs. How asthmatic lung fibroblasts respond to TNFα stimulation and subsequently regulate immune responses is not well understood. Endoplasmic reticulum (ER) stress and unfolded protein responses (UPR) play important roles in asthma, but their functional roles are still under investigation. In this study, we investigated TNFα-induced cytokine production in primary lung fibroblasts from asthmatic vs. non-asthmatic human subjects, and downstream effects on type 2 immune responses. TNFα significantly upregulated IL-6, IL-8, C-C motif chemokine ligand 5 (CCL5), and thymic stromal lymphopoietin (TSLP) mRNA expression and protein secretion by lung fibroblasts. Asthmatic lung fibroblasts secreted higher levels of TSLP which promoted IL-33-induced IL-5 and IL-13 production by peripheral blood mononuclear cells. TNFα exposure enhanced expression of ER stress/UPR pathways in both asthmatic and non-asthmatic lung fibroblasts, especially inositol-requiring protein 1α in asthmatics. ER stress/UPR inhibitors decreased IL-6, CCL5, and TSLP protein secretion by asthmatic lung fibroblasts. Our data suggest that TNFα and lung fibroblasts form an important axis in asthmatic lungs to promote asthmatic inflammation that can be attenuated by inhibiting ER stress/UPR pathway.

Lung function declines as people age and their lungs become stiffer. With an increasing elderly population, understanding mechanisms that contribute to these structural and functional changes in the aging lung is important. Part of the aging process is characterized by thicker, more fibrotic airways, and senile emphysema caused by changes in lung parenchyma. There is also senescence, which occurs throughout the body with aging. Here, using human airway smooth muscle (ASM) cells from patients in different age groups, we explored senescence pathways and changes in intracellular calcium signaling and extracellular matrix (ECM) deposition to elucidate potential mechanisms by which aging leads to thicker and stiffer lungs. Senescent markers p21, γH2AX, and β-gal, and some senescence-associated secretory proteins (SASP) increased with aging, as shown by staining and biochemical analyses. Agonist-induced intracellular Ca2+ responses, measured using fura-2 loaded cells and fluorescence imaging, increased with age. However, biochemical analysis showed that expression of the following markers decreased with age: M3 muscarinic receptor, TRPC3, Orai1, STIM1, SERCA2, MMP2 and MMP9. In contrast, collagen III, and fibronectin deposition increased with age. These data show that senescence increases in the aging airways that is associated with a stiffer but surprisingly greater intracellular calcium signaling as a marker for contractility. ASM senescence may enhance fibrosis in a feed forward loop promoting remodeling and altered calcium storage and buffering.

Background: Continuous positive airway pressure (CPAP) in preterm infants is initially beneficial, but animal models suggest longer term detrimental airway effects towards asthma. We used a neonatal CPAP mouse model and human fetal airway smooth muscle (ASM) to investigate the role of extracellular calcium-sensing receptor (CaSR) in these effects., Methods: Newborn wild type and smooth muscle-specific CaSR -/- mice were given CPAP for 7 days via a custom device (mimicking CPAP in premature infants), and recovered in normoxia for another 14 days (representing infants at 3-4 years). Airway reactivity was tested using lung slices, and airway CaSR quantified. Role of CaSR was tested using NPS2143 (inhibitor) or siRNA in WT mice. Fetal ASM cells stretched cyclically with/without static stretch mimicking breathing and CPAP were analyzed for intracellular Ca 2+ ([Ca 2+ ] i ) responses, role of CaSR, and signaling cascades., Results: CPAP increased airway reactivity in WT but not CaSR -/- mice, increasing ASM CaSR. NPS2143 or CaSR siRNA reversed CPAP effects in WT mice. CPAP increased fetal ASM [Ca 2+ ] I , blocked by NPS2143, and increased ERK1/2 and RhoA suggesting two mechanisms by which stretch increases CaSR., Conclusions: These data implicate CaSR in CPAP effects on airway function with implications for wheezing in former preterm infants., Impact: Neonatal CPAP increases airway reactivity to bronchoconstrictor agonist. CPAP increases smooth muscle expression of the extracellular calcium-sensing receptor (CaSR). Inhibition or absence of CaSR blunts CPAP effects on contractility. These data suggest a causal/contributory role for CaSR in stretch effects on the developing airway. These data may impact clinical recognition of the ways that CPAP may contribute to wheezing disorders of former preterm infants., (© 2021. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Supplemental O 2 (hyperoxia; 30-90% O 2 ) is a necessary intervention for premature infants, but it contributes to development of neonatal and pediatric asthma, necessitating better understanding of contributory mechanisms in hyperoxia-induced changes to airway structure and function. In adults, environmental stressors promote formation of senescent cells that secrete factors (senescence-associated secretory phenotype), which can be inflammatory and have paracrine effects that enhance chronic lung diseases. Hyperoxia-induced changes in airway structure and function are mediated in part by effects on airway smooth muscle (ASM). In the present study, using human fetal ASM cells as a model of prematurity, we ascertained the effects of clinically relevant moderate hyperoxia (40% O 2 ) on cellular senescence. Fetal ASM exposed to 40% O 2 for 7 days exhibited elevated concentrations of senescence-associated markers, including β-galactosidase; cell cycle checkpoint proteins p16, p21, and p-p53; and the DNA damage marker p-γH2A.X (phosphorylated γ-histone family member X). The combination of dasatinib and quercetin, compounds known to eliminate senescent cells (senolytics), reduced the number of hyperoxia-exposed β-galactosidase-, p21-, p16-, and p-γH2A.X-positive ASM cells. The senescence-associated secretory phenotype profile of hyperoxia-exposed cells included both profibrotic and proinflammatory mediators. Naive ASM exposed to media from hyperoxia-exposed senescent cells exhibited increased collagen and fibronectin and higher contractility. Our data show that induction of cellular senescence by hyperoxia leads to secretion of inflammatory factors and has a functional effect on naive ASM. Cellular senescence in the airway may thus contribute to pediatric airway disease in the context of sequelae of preterm birth.