Small airway fibroblasts from patients with chronic obstructive pulmonary disease exhibit cellular senescence.

Small airway disease (SAD) is a key early-stage pathology of chronic obstructive pulmonary disease (COPD). COPD is associated with cellular senescence whereby cells undergo growth arrest and express the senescence-associated secretory phenotype (SASP) leading to chronic inflammation and tissue remodeling. Parenchymal-derived fibroblasts have been shown to display senescent properties in COPD, however small airway fibroblasts (SAFs) have not been investigated. Therefore, this study investigated the role of these cells in COPD and their potential contribution to SAD. To investigate the senescent and fibrotic phenotype of SAF in COPD, SAFs were isolated from nonsmoker, smoker, and COPD lung resection tissue (n = 9-17 donors). Senescence and fibrotic marker expressions were determined using iCELLigence (proliferation), qPCR, Seahorse assay, and ELISAs. COPD SAFs were further enriched for senescent cells using FACSAria Fusion based on cell size and autofluorescence (10% largest/autofluorescent vs. 10% smallest/nonautofluorescent). The phenotype of the senescence-enriched population was investigated using RNA sequencing and pathway analysis. Markers of senescence were observed in COPD SAFs, including senescence-associated ß-galactosidase, SASP release, and reduced proliferation. Because the pathways driving this phenotype were unclear, we used cell sorting to enrich senescent COPD SAFs. This population displayed increased p21CIP1 and p16INK4a expression and mitochondrial dysfunction. RNA sequencing suggested these senescent cells express genes involved in oxidative stress response, fibrosis, and mitochondrial dysfunction pathways. These data suggest COPD SAFs are senescent and may be associated with fibrotic properties and mitochondrial dysfunction. Further understanding of cellular senescence in SAFs may lead to potential therapies to limit SAD progression.NEW & NOTEWORTHY Fibroblasts and senescence are thought to play key roles in the pathogenesis of small airway disease and COPD; however, the characteristics of small airway-derived fibroblasts are not well explored. In this study we isolate and enrich the senescent small airway-derived fibroblast (SAF) population from COPD lungs and explore the pathways driving this phenotype using bulk RNA-seq.

Talin 1 and 2 are required for myoblast fusion, sarcomere assembly and the maintenance of myotendinous junctions.

Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, talin 1 regulates the stability of myotendinous junctions (MTJs), but the function of talin 2 in skeletal muscle is not known. Here we show that MTJ integrity is affected in talin 2-deficient mice. Concomitant ablation of talin 1 and 2 leads to defects in myoblast fusion and sarcomere assembly, resembling defects in muscle lacking beta1 integrins. Talin 1/2-deficient myoblasts express functionally active beta1 integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather by disruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesion complexes is perturbed in the remaining muscle fibers of talin 1/2-deficient mice. We conclude that talin 1 and 2 are crucial for skeletal muscle development, where they regulate myoblast fusion, sarcomere assembly and the maintenance of MTJs.

Progressive myopathy and defects in the maintenance of myotendinous junctions in mice that lack talin 1 in skeletal muscle.

The development and function of skeletal muscle depend on molecules that connect the muscle fiber cytoskeleton to the extracellular matrix (ECM). beta1 integrins are ECM receptors in skeletal muscle, and mutations that affect the alpha7beta1 integrin cause myopathy in humans. In mice, beta1 integrins control myoblast fusion, the assembly of the muscle fiber cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The effector molecules that mediate beta1 integrin functions in muscle are not known. Previous studies have shown that talin 1 controls the force-dependent assembly of integrin adhesion complexes and regulates the affinity of integrins for ligands. Here we show that talin 1 is essential in skeletal muscle for the maintenance of integrin attachment sites at MTJs. Mice with a skeletal muscle-specific ablation of the talin 1 gene suffer from a progressive myopathy. Surprisingly, myoblast fusion and the assembly of integrin-containing adhesion complexes at costameres and MTJs advance normally in the mutants. However, with progressive ageing, the muscle fiber cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles show defects in the ability of talin 1-deficient muscle to generate force. Collectively, our findings show that talin 1 is essential for providing mechanical stability to integrin-dependent adhesion complexes at MTJs, which is crucial for optimal force generation by skeletal muscle.

Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo.

Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affinity state for their ligands, and they shift to a high affinity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin beta subunits is necessary and sufficient to trigger the activation of integrins to this high affinity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talin-deficient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.