Grp78 Loss in Epithelial Progenitors Reveals an Age-linked Role for Endoplasmic Reticulum Stress in Pulmonary Fibrosis.

Rationale: Alveolar epithelial cell (AEC) injury and dysregulated repair are implicated in the pathogenesis of pulmonary fibrosis. Endoplasmic reticulum (ER) stress in AEC has been observed in idiopathic pulmonary fibrosis (IPF), a disease of aging.Objectives: To investigate a causal role for ER stress in the pathogenesis of pulmonary fibrosis (PF) and therapeutic potential of ER stress inhibition in PF.Methods: The role of ER stress in AEC dysfunction and fibrosis was studied in mice with tamoxifen (Tmx)-inducible deletion of ER chaperone Grp78, a key regulator of ER homeostasis, in alveolar type II (AT2) cells, progenitors of distal lung epithelium, and in IPF lung slice cultures.Measurements and Main Results:Grp78 deletion caused weight loss, mortality, lung inflammation, and spatially heterogeneous fibrosis characterized by fibroblastic foci, hyperplastic AT2 cells, and increased susceptibility of old and male mice, all features of IPF. Fibrosis was more persistent in more severely injured Grp78 knockout (KO) mice. Grp78 KO AT2 cells showed evidence of ER stress, apoptosis, senescence, impaired progenitor capacity, and activation of TGF-ß (transforming growth factor-ß)/SMAD signaling. Glucose-regulated protein 78 is reduced in AT2 cells from old mice and patients with IPF, and ER stress inhibitor tauroursodeoxycholic acid ameliorates ER stress and fibrosis in Grp78 KO mouse and IPF lung slice cultures.Conclusions: These results support a causal role for ER stress and resulting epithelial dysfunction in PF and suggest ER stress as a potential mechanism linking aging to IPF. Modulation of ER stress and chaperone function may offer a promising therapeutic approach for pulmonary fibrosis.

Establishment of the early cilia preassembly protein complex during motile ciliogenesis.

Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Förster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.

Application of iPSC to Modelling of Respiratory Diseases.

Respiratory disease is one of the leading causes of morbidity and mortality world-wide with an increasing incidence as the aged population prevails. Many lung diseases are treated for symptomatic relief, with no cure available, indicating a critical need for novel therapeutic strategies. Such advances are hampered by a lack of understanding of how human lung pathologies initiate and progress. Research on human lung disease relies on the isolation of primary cells from explanted lungs or the use of immortalized cells, both are limited in their capacity to represent the genomic and phenotypic variability among the population. In an era where we are progressing toward precision medicine the use of patient specific induced pluripotent cells (iPSC) to generate models, where sufficient primary cells and tissues are scarce, has increased our capacity to understand human lung pathophysiology. Directed differentiation of iPSC toward lung presented the initial challenge to overcome in generating iPSC-derived lung epithelial cells. Since then major advances have been made in defining protocols to specify and isolate specific lung lineages, with the generation of airway spheroids and multi cellular organoids now possible. This technological advance has opened up our capacity for human lung research and prospects for autologous cell therapy. This chapter will focus on the application of iPSC to studying human lung disease.

Paediatric medial humeral epicondyle fracture management: 2019 approach.

PURPOSE OF REVIEW: The management of displaced medial humeral epicondyle fractures in children remains controversial. The indications for surgery, the ideal surgical strategy and the implications of a painful nonunion remain unclear. RECENT FINDINGS: This article describes the state of the evidence and the art in the management of medial humeral epicondyle fractures concentrating on recent research and current opinion. Treatment of paediatric medial epicondylar fractures of the elbow remains the domain of expert opinion and subject to great variance. Anatomical, biomechanical and computer simulation models suggest great importance should be given to the medial epicondyle and the structures, which insert onto it. However, this does not correlate with outcomes as reported by patients, parents and surgeons. SUMMARY: The question of which paediatric medial humeral epicondylar fractures benefit from operative fixation remains unanswered. A large randomized prospective trial is required.

CLDN18.1 attenuates malignancy and related signaling pathways of lung adenocarcinoma in vivo and in vitro.

Claudins are a family of transmembrane proteins integral to the structure and function of tight junctions (TJ). Disruption of TJ and alterations in claudin expression are important features of invasive and metastatic cancer cells. Expression of CLDN18.1, the lung-specific isoform of CLDN18, is markedly decreased in lung adenocarcinoma (LuAd). Furthermore, we recently observed that aged Cldn18 -/- mice have increased propensity to develop LuAd. We now demonstrate that CLDN18.1 expression correlates inversely with promoter methylation and with LuAd patient mortality. In addition, when restored in LuAd cells that have lost expression, CLDN18.1 markedly attenuates malignant properties including xenograft tumor growth in vivo as well as cell proliferation, migration, invasion and anchorage-independent colony formation in vitro. Based on high throughput analyses of Cldn18 -/- murine lung alveolar epithelial type II cells, as well as CLDN18.1-repleted human LuAd cells, we hypothesized and subsequently confirmed by Western analysis that CLDN18.1 inhibits insulin-like growth factor-1 receptor (IGF-1R) and AKT phosphorylation. Consistent with recent data in Cldn18 -/- knockout mice, expression of CLDN18.1 in human LuAd cells also decreased expression of transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) and their target genes, contributing to its tumor suppressor activity. Moreover, analysis of LuAd cells in which YAP and/or TAZ are silenced with siRNA suggests that inhibition of TAZ, and possibly YAP, is also involved in CLDN18.1-mediated AKT inactivation. Taken together, these data indicate a tumor suppressor role for CLDN18.1 in LuAd mediated by a regulatory network that encompasses YAP/TAZ, IGF-1R and AKT signaling.

Blockade of voltage-dependent K+ current in rabbit coronary arterial smooth muscle cells by the tricyclic antidepressant clomipramine.

We investigated the effect of the tricyclic antidepressant clomipramine on voltage-dependent K+ (Kv) channels in native rabbit coronary arterial smooth muscle cells. Our results showed that clomipramine inhibited vascular Kv channels in a concentration-dependent manner, with an IC50 value of 8.61 ± 4.86 µM and a Hill coefficient (n) of 0.58 ± 0.07. The application of 10 µM clomipramine did not affect the activation curves of the Kv channels; however, the inactivation curves of the Kv channels were shifted toward a more negative potential. The clomipramine-induced inhibition of Kv currents was not changed by the application of train pulses (1 or 2 Hz), which demonstrated that clomipramine inhibited Kv current in a state (use)-independent manner. Pretreatment with the Kv1.5 and Kv2.1 inhibitors, DPO-1 and guangxitoxin, respectively, partially reduced the clomipramine-induced inhibition of Kv currents. Therefore, we concluded that clomipramine inhibited vascular Kv channels in a concentration-dependent, but state (use)-independent manner, regardless of its own function.

Embryonic stem cell potency fluctuates with endogenous retrovirus activity.

Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.

Inhibitory effect of the tricyclic antidepressant amitriptyline on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

Amitriptyline, a tricyclic antidepressant (TCA) drug, is widely used in treatment of psychiatric disorders. However, the side effects of amitriptyline on vascular K+ channels remain to be determined. Therefore, we investigated the effect of the tricyclic antidepressant and serotonin reuptake inhibitor amitriptyline on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells, using the whole-cell patch clamp technique. The Kv current amplitudes were inhibited by amitriptyline in a concentration-dependent manner, with an apparent IC50 value of 2.2 ± 0.14 µmol/L and a Hill coefficient of 0.87 ± 0.03. Amitriptyline shifted the activation curve to a more positive potential, but had no significant effect on the inactivation curve, suggesting that amitriptyline altered the voltage sensitivity of Kv channels. Pretreatment with Kv1.5 and Kv1.2 channel inhibitors did not alter the inhibitory effect of amitriptyline on Kv channels. Additionally, application of train pulses (1 and 2 Hz) did not affect amitriptyline-induced inhibition of Kv currents, which suggested that the action of amitriptyline on Kv channels was not use (state)-dependent. From these results, we concluded that amitriptyline inhibited the channels in a concentration-dependent, but state-independent manner.

Inhibition of the voltage-dependent K+ current by the class Ic antiarrhythmic drug flecainide in rabbit coronary arterial smooth muscle cells.

This study examined the inhibitory effect of flecainide, a class 1c antiarrhythmic agent (Na+ channel blocker), on voltage-dependent K+ (Kv) channels in smooth muscle cells isolated from coronary arteries. Flecainide decreased the vascular Kv channel current in a dose-dependent manner with an IC50 value of 5.90 ± 0.87 µmol/L and a Hill coefficient of 0.77 ± 0.06. Although the steady-state activation curve was not affected by flecainide, it shifted the steady-state inactivation curves toward a more negative potential. Application of train pulses such as 1 or 2 Hz did not change the flecainide-induced inhibition of Kv channels, indicating that the inhibitory effect of flecainide was not use-dependent. Using perforated-patch clamp experiments, we found that inhibition of Kv channels by flecainide caused membrane depolarization. Together, these results suggest that flecainide inhibits Kv channels in a concentration-dependent, but not use-dependent manner by changing the inactivation gating properties. Furthermore, Kv channel inhibition by flecainide occurs regardless of Na+ channel inhibition.

The selective serotonin reuptake inhibitor dapoxetine inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

We investigated the inhibitory effect of dapoxetine, a selective serotonin reuptake inhibitor (SSRI), on voltage-dependent K+ (Kv) channels using native smooth muscle cells from rabbit coronary arteries. Dapoxetine inhibited Kv channel currents in a concentration-dependent manner, with an IC50 value of 2.68±0.94 µmol/L and a slope value (Hill coefficient) of 0.63±0.11. Application of 10 µmol/L dapoxetine accelerated the rate of inactivation of Kv currents. Although dapoxetine did not modify current activation kinetics, it caused a significant negative shift in the inactivation curves. Application of train step (1 or 2 Hz) progressively increased the inhibitory effect of dapoxetine on Kv channels. In addition, the recovery time constant was extended in its presence, suggesting that the longer recovery time constant from inactivation underlies a use-dependent inhibition of the channel. From these results, we conclude that dapoxetine inhibits Kv channels in a dose-, time-, use-, and state (open)-dependent manner, independent of serotonin reuptake inhibition.

Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells.

Despite therapeutic advancement, pulmonary disease still remains a major cause of morbidity and mortality around the world. Opportunities to study human lung disease either in vivo or in vitro are currently limited. Using induced pluripotent stem cells (iPSCs), we generated mature multiciliated cells in a functional airway epithelium. Robust multiciliogenesis occurred when notch signaling was inhibited and was confirmed by (i) the assembly of multiple pericentrin-stained centrioles at the apical surface, (ii) expression of transcription factor forkhead box protein J1, and (iii) presence of multiple acetylated tubulin-labeled cilia projections in individual cells. Clara, goblet, and basal cells were all present, confirming the generation of a complete polarized epithelial-cell layer. Additionally, cAMP-activated and cystic fibrosis transmembrane regulator inhibitor 172-sensitive cystic fibrosis transmembrane regulator currents were recorded in isolated epithelial cells. Our report demonstrating the generation of mature multiciliated cells in respiratory epithelium from iPSCs is a significant advance toward modeling a number of human respiratory diseases in vitro.

Nortriptyline, a tricyclic antidepressant, inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells.

We demonstrated the effect of nortriptyline, a tricyclic antidepressant drug and serotonin reuptake inhibitor, on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Nortriptyline inhibited Kv currents in a concentration-dependent manner, with an apparent IC50 value of 2.86±0.52 µM and a Hill coefficient of 0.77±0.1. Although application of nortriptyline did not change the activation curve, nortriptyline shifted the inactivation current toward a more negative potential. Application of train pulses (1 or 2 Hz) did not change the nortriptyline-induced Kv channel inhibition, suggesting that the effects of nortiprtyline were not use-dependent. Preincubation with the Kv1.5 and Kv2.1/2.2 inhibitors, DPO-1 and guangxitoxin did not affect nortriptyline inhibition of Kv channels. From these results, we concluded that nortriptyline inhibited Kv channels in a concentration-dependent and state-independent manner independently of serotonin reuptake.

Alterations of voltage-dependent K(+) channels in the mesenteric artery during the early and chronic phases of diabetes.

This study investigated the alteration of voltage-dependent K(+) (Kv) channels in mesenteric arterial smooth muscle cells from control (Long-Evans Tokushima Otsuka [LETO]) and diabetic (Otsuka Long-Evans Tokushima Fatty [OLETF]) rats during the early and chronic phases of diabetes. We demonstrated alterations in the mesenteric Kv channels during the early and chronic phase of diabetes using the patch-clamp technique, the arterial tone measurement system, and RT-PCR in Long-Evans Tokushima (LETO; for control) and Otsuka Long-Evans Tokushima Fatty (OLETF; for diabetes) type 2 diabetic model rats. In the early phase of diabetes, the amplitude of mesenteric Kv currents induced by depolarizing pulses was greater in OLETF rats than in LETO rats. The contractile response of the mesenteric artery induced by the Kv inhibitor, 4-aminopyridine (4-AP), was also greater in OLETF rats. The expression of most Kv subtypes- including Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.2, Kv4.1, Kv4.3, Kv5.1, Kv6.2, Kv8.1, Kv9.3, and Kv10.1-were increased in mesenteric arterial smooth muscle from OLETF rats compared with LETO rats. However, in the chronic phase of diabetes, the Kv current amplitude did not differ between LETO and OLETF rats. In addition, the 4-AP-induced contractile response of the mesenteric artery and the expression of Kv subtypes did not differ between the two groups. The increased Kv current amplitude and Kv channel-related contractile response were attributable to the increase in Kv channel expression during the early phase of diabetes. The increased Kv current amplitude and Kv channel-related contractile response were reversed during the chronic phase of diabetes.

Y-27632, a Rho-Associated Protein Kinase Inhibitor, Inhibits Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells.

We examined the effects of the Rho-associated protein kinase (ROCK) inhibitor Y-27632 on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch clamp technique. Y-27632 reduced the amplitude of the Kv current in a concentration-dependent manner, with an IC50 of 0.87 ± 0.06 µmol/l and a Hill coefficient of 1.48 ± 0.06. Y-27632 did not affect the steady-state activation or inactivation curves, suggesting that the drug does not affect the voltage sensitivity of Kv channels. Another ROCK inhibitor, H-1152, did not affect the Kv current and had no significant effect on the Y-27632-induced inhibition of Kv channels, indicating that the inhibitory effect of Y-27632 on the Kv current is independent of ROCK signaling. From these results, we conclude that Y-27632 inhibits the Kv channel current in a dose-dependent and ROCK signaling-independent manner.

The Effects of the Selective Serotonin Reuptake Inhibitor Fluvoxamine on Voltage-Dependent K(+) Channels in Rabbit Coronary Arterial Smooth Muscle Cells.

We demonstrated the inhibitory effect of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), on voltage-dependent K(+) (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Fluvoxamine reduced the amplitude of Kv currents in a concentration-dependent manner with an IC50 value of 3.71±1.09 µM and a Hill coefficient of 0.62±0.14. Although fluvoxamine did not significantly affect the steady-state activation curve, it shifted the steady-state inactivation curve toward a more negative potential. Pretreatment with another SSRI, paroxetine, did not affect the basal Kv current and did not alter the inhibitory effect of fluvoxamine on Kv channels. We concluded that fluvoxamine inhibits the Kv current in a concentration-dependent manner and in a closed (inactivated) state of the Kv channels independent of serotonin reuptake inhibition.

Postoperative Continuous Passive Motion Does Not Improve the Range of Movement Achieved After Manipulation Under Anesthetic for Stiffness in Total Knee Replacement.

Background: Stiffness is a common complication following total knee arthroplasty. Manipulation under anesthesia (MUA) is an intervention that can potentially improve range of motion (ROM). Continuous passive motion (CPM) therapy has been utilized to enhance post-MUA ROM, but its effectiveness remains debated. This study assesses whether CPM therapy after MUA results in superior ROM outcomes compared to MUA alone. Methods: A retrospective analysis included patients undergoing MUA for stiff primary total knee arthroplasty between 2017 and 2022. Demographics and ROM data were collected. Patients were in 2 groups: those who received inpatient CPM post-MUA and those who received day-case MUA alone. Complications and further interventions were noted. Results: Of 126 patients, 39 underwent MUA only (day-case group), and 87 received CPM and MUA (inpatient group). Mean preoperative ROM was 69.4° (standard deviation [SD]:18.0°) and 73.9° (SD: 18.1°) for inpatient and day-case groups, respectively. Mean post-MUA ROM improved by 39.4° (SD: 17.7°) and 25.5° (SD: 11.1°) inpatient groups and day-case, respectively. The mean percentage of ROM gained at MUA maintained at final follow-up was 63.7% (40.8%) and 67.0% (47.5%) inpatient and day-case groups, respectively. Conclusions: This study found no advantage in the routine use of CPM post-MUA for stiff total knee replacement patients, suggesting it may not provide sustained ROM improvements compared to MUA alone. Cost-effectiveness and patient selection merit further investigation.

Regulation of ca(2+) signaling in pulmonary hypertension.

Understanding the cellular and molecular mechanisms involved in the development and progression of pulmonary hypertension (PH) remains imperative if we are to successfully improve the quality of life and life span of patients with the disease. A whole plethora of mechanisms are associated with the development and progression of PH. Such complexity makes it difficult to isolate one particular pathway to target clinically. Changes in intracellular free calcium concentration, the most common intracellular second messenger, can have significant impact in defining the pathogenic mechanisms leading to its development and persistence. Signaling pathways leading to the elevation of [Ca(2+)](cyt) contribute to pulmonary vasoconstriction, excessive proliferation of smooth muscle cells and ultimately pulmonary vascular remodeling. This current review serves to summarize the some of the most recent advances in the regulation of calcium during pulmonary hypertension.

PDGF enhances store-operated Ca2+ entry by upregulating STIM1/Orai1 via activation of Akt/mTOR in human pulmonary arterial smooth muscle cells.

Platelet-derived growth factor (PDGF) and its receptor are known to be substantially elevated in lung tissues and pulmonary arterial smooth muscle cells (PASMC) isolated from patients and animals with pulmonary arterial hypertension. PDGF has been shown to phosphorylate and activate Akt and mammalian target of rapamycin (mTOR) in PASMC. In this study, we investigated the role of PDGF-mediated activation of Akt signaling in the regulation of cytosolic Ca(2+) concentration and cell proliferation. PDGF activated the Akt/mTOR pathway and, subsequently, enhanced store-operated Ca(2+) entry (SOCE) and cell proliferation in human PASMC. Inhibition of Akt attenuated the increase in cytosolic Ca(2+) concentration due to both SOCE and PASMC proliferation. This effect correlated with a significant downregulation of stromal interacting molecule (STIM) and Orai, proposed molecular correlates for SOCE in many cell types. The data from this study present a novel pathway for the regulation of Ca(2+) signaling and PASMC proliferation involving activation of Akt in response to upregulated expression of PDGF. Targeting this pathway may lead to the development of a novel therapeutic option for the treatment of pulmonary arterial hypertension.

Brief report: efficient generation of hematopoietic precursors and progenitors from human pluripotent stem cell lines.

By mimicking embryonic development of the hematopoietic system, we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines, extra cellular matrix components, and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% ± 16%, from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability.