Citations

Stress failure of alveolar epithelial cells is a common feature of ventilator‐injured lungs. While some injured cells undergo necrosis, others repair their plasma membrane (PM) wounds through non‐secretory exocytosis of intracellular vesicles (e.g. lysosomes) to and cytoskeleton remodeling at and around the wound. We measured the timing of lysosomal trafficking and F‐actin reorganization following micropuncture injury in primary type I alveolar epithelial cells (ATI). ATI were isolated from female rats and loaded with Calcein AM, N‐rhodamine phosphatidylethanolamine (lysosome membrane label) or transfected with LifeAct (GFP tagged actin label). Changes in fluorescence intensity were indicators of time to wound repair (cessation of calcein fluorescence decay following injury), lysosomal trafficking to the wound site, and actin remodeling. ATI repaired wounds within 27.9±3.9s. Recruitment of lysosomes to the wound site was detected at 2.9±1.5s following injury and preceded actin polymerization by 23.7±5.4s. Failure to repair PM wounds was associated with a weak or absent actin polymerization response. We conclude that local actin polymerization is a critical step in PM wound repair and postulate that adhesive interactions between PM lipids and the remodeled actin cytoskeleton serve as a driving force for wound closure by promoting lateral PM lipid bilayer flow. Support: HL63178

Regulation of intracellular Ca2+ in human airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca2+ release/reuptake and plasma membrane (PM) Ca2+ influx, including store‐operated Ca2+ entry (SOCE) following SR Ca2+ depletion. Studies in human ASM have shown that siRNA suppression of stromal interaction molecule 1 (STIM1) and Orai1 decrease SOCE; however, their exact role in regulating Ca2+ are still under investigation. In human ASM, STIM1 and STIM2 were found exclusively in SR membrane, and Orai1 only in PM (especially caveolae). GFP‐tagged STIM1 transfection showed intracellular/SR localization at baseline, with increased peri‐PM aggregation following store depletion with 10ƒÝM cyclopiazonic acid (CPA). Such aggregation was prevented by microtubule inhibition (nocodazole), but not actin inhibition (phalloidin). FRET analysis revealed co‐localization of STIM1 with TRPC3 and Orai1 in CPA‐stimulated cells. In fura‐2 loaded ASM cells, SOCE was significantly reduced by STIM1 and Orai1 siRNA (less so by STIM2 siRNA). However, basal Ca2+ and agonist (ACh, histamine)‐induced Ca2+ responses were substantially modulated by STIM2 suppression. These results indicate that in human ASM, STIM1 mediates SR‐PM interactions in regulating SOCE, while STIM2 modulates sub‐maximal agonist‐induced changes in Ca2+. Supported by NIH grant HL74309 (GCS), HL090595 (CMP) and HL088029 (YSP).

Plasma membrane Ca 2+ influx and sarcoplasmic reticulum (SR) Ca 2+ release are key components of intracellular Ca 2+ regulation in airway smooth muscle (ASM). We and others have previously found that ASM Ca 2+ regulation involves propagating Ca 2+ oscillations via these mechanisms. Ca 2+ oscillations in non‐excitable cells involve influx via arachidonic acid (AA) regulated channels (ARC); however, little is known about ARC in ASM. In human ASM cells loaded with fura2 (5 μM), we found that 10 μM AA triggered Ca 2+ oscillations. OAG, oleic acid and palmitic acid (10 μM) failed to elicit Ca 2+ responses. Pre‐incubation with LaCl 3 1 μM and 1mM; 5 min) inhibited AA‐induced oscillations. Inhibition of SR Ca 2+ release (ryanodine 10 μM and IP3 channel inhibitor Xestospongin C 10 μM; 30 min), decreased Ca 2+ oscillation frequency and amplitude. Inhibitors of receptor‐operated channels (SKF‐96365; 10 μM), lipoxygenase (zileuton; 10 μM), or cyclooxygenase (indomethacin; 1 μM), did not affect oscillations. Finally, siRNA for Caveolin‐1, STIM1 and Orai3 (25nM) reduced frequency and amplitude of AA‐induced oscillations. These results indicate a novel caveolar Ca 2+ influx mechanism in human ASM mediated via AA that interacts with SR to produce repetitive SR Ca 2+ release. Such AA‐mediated influx could potentially contribute to increased Ca 2+ in ASM during inflammation. Supported by NIH grants HL090595 (CMP) and HL088029 (YSP)

Enhanced airway contractility by inflammatory cytokines involves increased intracellular Ca 2+ levels in airway smooth muscle (ASM). In this study, we examined the role of the bidirectional plasma membrane Na + /Ca 2+ exchanger (NCX) in human ASM Ca 2+ regulation following exposure to the pro‐inflammatory cytokines tumor necrosis factor alpha (TNFα) or interleukin‐13 (IL‐13). Both cytokines significantly increased NCX1 expression, with these effects inhibited by dexamethasone (NFκB inhibitor) or PD98059 (MAPK inhibitor). In human ASM cells loaded with the Ca 2+ indicator fura‐2, or the intracellular Na + indicator SBFI, NCX‐mediated Ca 2+ influx (with simultaneous Na + efflux) was enhanced by both cytokines. NCX‐mediated Ca 2+ efflux (with simultaneous Na + influx) was also enhanced, but not to the same extent. The NCX inhibitor KBR7943 as well as NCX1 siRNA inhibited cytokine enhancement of both modes NCX and of Ca 2+ responses to bronchoconstrictor agonists. These data indicate that a) NCX normally operates in human ASM; b) cytokines increase NCX expression, leading to greater NCX‐mediated Ca 2+ influx, thus contributing to enhanced Ca 2+ in inflamed ASM. Supported by NIH grants HL74309 (GCS), HL090595‐S2 (VS) and HL090595 (CMP).

Senescent cells exacerbate COVID-19 Cellular senescence is a state elicited in response to stress signals and is associated with a damaging secretory phenotype. The number of senescent cells increases with advanced age and this in turn drives age-related diseases. Camell et al. show that senescent cells have an amplified inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (see the Perspective by Cox and Lord). This response is communicated to nonsenescent cells, suppressing viral defense mechanisms and increasing the expression of viral entry proteins. In old mice infected with a SARS-CoV-2–related virus, treatment with senolytics to reduce the senolytic cell burden reduced mortality and increased antiviral antibodies. Science , abe4832, this issue p. eabe4832 ; see also abi4474, p. 281

Differentiation of human bronchial epithelial cells (HBEs) in air-liquid interface (ALI) cultures recapitulates organotypic modeling of the in vivo environment. Although ALI cultures are invaluable for studying the respiratory epithelial barrier, loss-of-function studies are limited by potentially cytotoxic reagents in classical transfection methods, the length of the differentiation protocol, and the number of primary epithelial cell passages. Here, we present the efficacy and use of a simple method for small interfering RNA (siRNA) transfection of normal HBEs (NHBEs) in ALI cultures that does not require potentially cytotoxic transfection reagents and does not detrimentally alter the physiology or morphology of NHBEs during the differentiation process. This transfection protocol introduces a reproducible and efficient method for loss-of-function studies in HBE ALI cultures that can be leveraged for modeling the respiratory system and airway diseases.

Treatment resistant restless legs syndrome (RLS) in the setting of psychiatric comorbidities can be difficult to manage. Our patient is a 69-year-old Caucasian gentleman with bipolar disorder type I, unspecified anxiety disorder, obstructive sleep apnea (OSA), and treatment-refractory RLS. At initial presentation, the patient’s prescribed medication regimen included fluoxetine 40 mg daily, gabapentin 800 mg in the morning and 3200 mg at bedtime, pramipexole 0.375 mg daily, lamotrigine 200 mg daily, trazodone 200 mg at bedtime, and temazepam 15 to 30 mg as needed for insomnia and RLS. Over the course of nearly 4 years, treatment interventions for this patient’s RLS included: cognitive behavioral therapy for insomnia, discontinuation of exacerbating medications, switching dopamine agonists, use of pregabalin and iron supplement. This report demonstrates a challenging case of RLS in the setting of psychiatric comorbidities, development of augmentation, and polypharmacy.