Zissis C. Chroneos

Understanding the human distal lung requires ex-vivo models that capture both the structural hierarchy and mechanical environment of alveolar sacs, yet current ex-vivo systems fall short. Manually cultured organoids and two-dimensional cell cultures lack structural hierarchy, apical access, and physiologic actuation via ventilation, limiting their use in modeling infection and mechano-transduction. Here, we established three-dimensional (3D) alveolar epithelial sacculoids (AES) by bioprinting pluripotent stem cell-derived alveolar epithelial type II cells (ATIIs) into defined 3D geometries in high density. AES reproducibly self-organized into multi-unit, lumenized sacs with polarized epithelia, surfactant secretion, and functional heterogeneity including proliferative ATIIs, surfactant-producing ATIIs, and transitional pre-alveolar type I transitional cell state (PATS)-like cells. A custom air-driven platform enabled fluid-mediated 3D ventilation, producing volumetric oscillations across closed sacs. This actuation engaged canonical (Ser127) and non-canonical (Tyr357, integrin–FAK–dependent) Hippo signaling, driving ATII-to-ATI remodeling and junctional stabilization. Upon apical infection with influenza virus, AES recapitulated canonical antiviral responses and epithelial plasticity resembling injury-induced alveolar repair, including depletion of functional ATIIs and emergence of proliferative ATIIs and transitional states. AES represent a physiologically ventilated model of the human alveolar niche, enabling mechanistic studies of epithelial plasticity, viral pathogenesis, and biomechanical signaling under physiologically-relevant conditions. Overall, our model provides a foundation for future integration of stromal, vascular, and immune components toward full alveolar mimicry to facilitate ex-vivo translational respiratory research.

Triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options. TAS-115, a multi-receptor tyrosine kinase inhibitor, has not previously been evaluated in TNBC. Here, we investigated its therapeutic effects alone and in combination with doxorubicin (DOXO), using three-dimensional heterotypic spheroid models, including free-standing, bioprinted static, and perfused systems. TAS-115 significantly reduced cell proliferation and viability, enhanced apoptosis, and suppressed c-mesenchymal-epithelial transition/hepatocyte growth factor and PI3K/Akt/mTOR signaling. Combined treatment with DOXO further amplified these effects. In perfused bioprinted models, TAS-115 markedly inhibited tumor cell migration, highlighting its potential to limit metastatic behavior. These findings identify TAS-115 as a promising therapeutic strategy for TNBC, either as a monotherapy or in combination with chemotherapy.

Health effects of electronic cigarettes (ECs) remain unknown, despite their popularity. We have determined that ECs produce highly reactive free radicals that could potentially cause damage in exposed tissues, mainly lungs. Goal for this study was to investigate the short- and long-term effects of ECs in mouse lungs. We focused on evaluating lung functions, oxidative stress related markers, and lung injury following nose-only exposures in male and female mice after 4- and 12-week periods. The EC exposure was modeled in vivo using nose-only exposures to C57BL/6 mice. For all studies, E-liquid (60:40; PG:VG) aerosols were compared to sham (compressed air) and to very low non-nicotine cigarette smoke (CS) controls in both sexes. Oxidative stress biomarkers (GSH, 8-Isoprostane, REDD1, and pGSK3β) and their selected downstream (RPS6) as well as upstream (AKT) target proteins in addition to pH2AX were measured by Western blot analysis. Lung function in mice was assessed by flexiVent and the injury scores were calculated following lung histology. Changes in cytology were also observed in cytospins from bronchoalveolar lavage (BALF). The lung injury (LI) score following 12-week exposures was significantly higher with EC and CS in female mice. Higher cell counts in BALF were mainly observed in CS exposed males and females at 4 and 12 weeks. 8-Isoprostane levels were significantly higher in EC and CS exposed males at 12 weeks. pGSK3β/GSK3β was low in males and higher in female mice at 4 weeks, and this difference was more pronounced at 12 weeks in CS exposed mice. Some mice exposed to EC and CS also showed DNA damage, as measured by pH2AX/H2AX expression. Based on the LI score, ECs were placed in between compressed air and CS. Our results showed the differentially expressed inflammation and oxidative stress/damage-related pathways from in vivo exposures to EC aerosols vs CS that could be an effective strategy for identifying EC relevant biomarkers of exposure and potential harm.

Bronchopulmonary dysplasia (BPD) is a condition of poor alveolar formation that causes chronic breathing impairment in infants born prematurely. Preterm lungs lack surfactant and are vulnerable to oxidative injuries driving the development of BPD. Our recent studies reported that surfactant protein A (SP-A) genetic variants influence susceptibility to neonatal lung disease. SP-A modulates activation of alveolar macrophages and parturition onset in late gestation. We asked whether a lack of SP-A alters alveolarization in a mouse model of hyperoxia-induced BPD. SP-A-deficient and control newborn mice were exposed to either clinically relevant 60% O₂ hyperoxia or normoxia for 5–7 days. Alveolar formation was then assessed by mean linear intercept (MLI) and radial alveolar count (RAC) measurements in lung tissue sections. We report that the combination of SP-A deficiency and hyperoxia reduces alveolar growth compared to WT mice. The morphometric analysis of normoxic SP-A-deficient lungs showed lower RAC compared to controls, indicating reduced alveolar number. In the presence of hyperoxia, MLI was higher in SP-A-deficient lungs compared to controls. Differences were statistically significant for female pups. Spatial proteomic profiling of lung tissue sections showed that hyperoxia caused a 4-fold increase in the DNA damage marker γH2Ax in macrophages of SP-A-deficient lungs compared to normoxia. Our short report suggests an important role for SP-A in perinatal lung development and the protection of lung macrophages from oxidant injury. These studies warrant future investigation to discern the temporal interaction of SP-A, gender, oxidant injury, and lung macrophages in perinatal alveolar formation and development of BPD.

Background-Bronchopulmonary dysplasia (BPD) is a condition of poor alveolar formation that causes chronic breathing impairment in infants born prematurely. Preterm lungs lack surfactant and are vulnerable to oxidative injuries driving the development of BPD. Our recent studies reported that surfactant protein A (SP-A) genetic variants influence susceptibility to neonatal lung disease. SP-A modulates activation of alveolar macrophages and parturition onset in late gestation. We asked whether a lack of SP-A alters alveolarization in a mouse model of hyperoxia-induced BPD. Methods- SP-A-deficient and control newborn mice were exposed to either clinically relevant 60% O2 hyperoxia or normoxia for 5-7 days. Alveolar formation was then assessed by mean linear intercept (MLI) and radial alveolar count (RAC) measurements in lung tissue sections. Results- We report that the combination of SP-A deficiency and hyperoxia reduces alveolar growth compared to WT mice. The morphometric analysis of normoxic SP-A-deficient lungs showed lower RAC compared to controls, indicating reduced alveolar number. In the presence of hyperoxia, MLI was higher in SP-A-deficient lungs compared to controls. Differences were statistically significant for female pups. Spatial proteomic profiling of lung tissue sections showed that hyperoxia caused a 4-fold increase in the DNA damage marker gammaH2Ax in macrophages of SP-A-deficient lungs compared to normoxia. Conclusions- Our short report suggests an important role for SP-A in perinatal lung development and in protecting lung macrophages from oxidant injury. These studies warrant future investigation to discern the temporal interaction of SP-A, gender, oxidant injury, and lung macrophages in perinatal alveolar formation and development of BPD.

Alternative and combinatorial splicing of myosin 18A ( MYO18A ) gene transcripts results in expression of MYO18A protein isoforms and isoform variants with different membrane and subcellular localizations, and functional properties. MYO18A proteins are members of the myosin superfamily consisting of a myosin‐like motor domain, an IQ motif, and a coiled‐coil domain. MYO18A isoforms, however, lack the ability to hydrolyze ATP and do not perform ATP‐dependent motor activity. MYO18A isoforms are distinguished by different amino‐ and carboxy‐terminal extensions and domains. The domain organization and functions of MYO18Aα, MYO18Aβ, and MYO18Aγ have been studied experimentally. MYO18Aα and MYO18Aβ have a common carboxy‐terminal extension but differ by the presence or absence of an amino‐terminal KE repeat and PDZ domain, respectively. The amino‐ and carboxy‐terminal extensions of MYO18Aγ contain unique proline and serine‐rich domains. Computationally predicted MYO18Aε and MYO18Aδ isoforms contain the carboxy‐terminal serine‐rich extension but differ by the presence or absence of the amino‐terminal KE/PDZ extension. Additional isoform variants within each category arise by alternative utilization or inclusion/exclusion of small exons. MYO18Aα variants are expressed in somatic cells and mature immune cells, whereas MYO18Aβ variants occur mainly in myeloid and natural killer cells. MYO18Aγ expression is selective to cardiac and skeletal muscle. In the present review perspective, we discuss current and emerging concepts of the functional specialization of MYO18A proteins in membrane and cytoskeletal dynamics, cellular communication and signaling, endocytic and exocytic organelle movement, viral infection, and as the SP‐R210 receptor for surfactant protein A.

To our knowledge, this study is the first to report that SP-A prevents delay of labor and inflammation-induced stillbirth through the receptor SP-R210_L.