Progressive fibrosing interstitial lung diseases (PFILDs) cause substantial morbidity and mortality. Antifibrotic agents slow progression, but most of the clinical need remains unmet. The archetypal PFILD is idiopathic pulmonary fibrosis (IPF). Chronic progression is driven by transforming growth factor (TGF-)β1 signalling. It is punctuated by inflammatory flares known as acute exacerbations (AE-IPF), which are associated with accelerated decline and high mortality. We hypothesized that acute injury responses underlying exacerbations and the mechanisms of chronic fibrosis overlap at the molecular level, via a cell surface assembly nucleated by galectin-3 that we term the ‘gal-3-fibrosome’. We focused upon a putative pro-inflammatory galectin-3 ligand, the CD98:integrin complex. Our data indicate CD98 and β1-integrin co-localise with galectin-3 within epithelial cells in IPF lung tissue, and within 40 nm in human lung tissue treated with TGF-β1 compared to controls. CD98 is required for interleukin (IL-)6 and IL-8 responses to biochemical and biophysical conditions mimicking stimuli of AE-IPF in vivo , ex vivo and in cells, and for an interstitial neutrophilic response in a mouse model. We demonstrate this pathway progresses via intracellular influx of Ca 2+ mediated by TRPV4, and NF-κB activation, operating in positive feedback. Lastly we show the CD98- and galectin-3-dependence of IL-6 and IL-8 responses to the SARS-CoV-2 spike protein receptor binding domain and the conservation of this response pattern between lung epithelial cells and monocyte-derived macrophages. Taken together our findings identify CD98 as a key mediator of both pro-fibrotic and acute inflammatory responses in the lung with relevance to AE- and chronic progression of IPF, and the priming of fibrotic lungs for acute inflammatory responses. They similarly implicate CD98 and galectin-3 as mediators of COVID pneumonitis and worse outcomes in ILD patients with COVID.

Physiological and premature aging are frequently associated with an accumulation of prelamin A, a precursor of lamin A, in the nuclear envelope of various cell types. Here, we aimed to underpin the hitherto unknown mechanisms by which prelamin A alters myonuclear organization and muscle fiber function. By experimentally studying membrane-permeabilized myofibers from various transgenic mouse lines, our results indicate that, in the presence of prelamin A, the abundance of nuclei and myosin content is markedly reduced within muscle fibers. This leads to a concept by which the remaining myonuclei are very distant from each other and are pushed to function beyond their maximum cytoplasmic capacity, ultimately inducing muscle fiber weakness.

The role of reactive oxygen species (ROS) in smooth muscle contraction is poorly understood. We hypothesised that G-protein coupled receptor (GPCR) activation and hypoxia induce Rho-kinase activity and contraction in rat intra-pulmonary artery (IPA) via stimulation of ROS production and subsequent Src-family kinase (SrcFK) activation. The T-type prostanoid receptor agonist U46619 induced ROS production in pulmonary artery smooth muscle cells (PASMC). U46619 also induced c-Src cysteine oxidation, SrcFK auto-phosphorylation, MYPT-1 and MLC20 phosphorylation and contraction in IPA, and all these responses were inhibited by antioxidants (ebselen, Tempol). Contraction and SrcFK/MYPT-1/MLC20 phosphorylations were also inhibited by combined superoxide dismutase and catalase, or by the SrcFK antagonist PP2, while contraction and MYPT-1/MLC20 phosphorylations were inhibited by the Rho guanine nucleotide exchange factor (RhoGEF) inhibitor Y16. H2O2 and the superoxide-generating quinoledione LY83583 both induced c-Src oxidation, SrcFK auto-phosphorylation and contraction in IPA. LY83583 and H2O2-induced contractions were inhibited by PP2, while LY83583-induced contraction was also inhibited by antioxidants and Y16. SrcFK auto-phosphorylation and MYPT-1/MLC20 phosphorylation was also induced by hypoxia in IPA and this was blocked by mitochondrial inhibitors rotenone and myxothiazol. In live PASMC, sub-cellular translocation of RhoA and the RhoGEF ARHGEF1 was triggered by both U46619 and LY83583 and this translocation was blocked by antioxidants and PP2. RhoA translocation was also inhibited by an ARHGEF1 siRNA. U46619 enhanced ROS-dependent co-immunoprecipitation of ARHGEF1 with c-Src. Our results demonstrate a link between GPCR-induced cytosolic ROS or hypoxia-induced mitochondrial ROS and SrcFK activity, Rho-kinase activity and contraction. ROS and SrcFK activate RhoA via ARHGEF1.

Application of H2S (“sulfide”) elicits a complex contraction in rat pulmonary arteries (PAs) comprising a small transient contraction (phase 1; Ph1) followed by relaxation and then a second, larger, and more sustained contraction (phase 2; Ph2). We investigated the mechanisms causing this response using isometric myography in rat second-order PAs, with Na2S as a sulfide donor. Both phases of contraction to 1,000 μM Na2S were attenuated by the pan-PKC inhibitor Gö6983 (3 μM) and by 50 μM ryanodine; the Ca2+ channel blocker nifedipine (1 μM) was without effect. Ph2 was attenuated by the mitochondrial complex III blocker myxothiazol (1 μM), the NADPH oxidase (NOX) blocker VAS2870 (10 μM), and the antioxidant TEMPOL (3 mM) but was unaffected by the complex I blocker rotenone (1 μM). The bath sulfide concentration, measured using an amperometric sensor, decreased rapidly following Na2S application, and the peak of Ph2 occurred when this had fallen to ~50 μM. Sulfide caused a transient increase in NAD(P)H autofluorescence, the offset of which coincided with development of the Ph2 contraction. Sulfide also caused a brief mitochondrial hyperpolarization (assessed using tetramethylrhodamine ethyl ester), followed immediately by depolarization and then a second more prolonged hyperpolarization, the onset of which was temporally correlated with the Ph2 contraction. Sulfide application to cultured PA smooth muscle cells increased reactive oxygen species (ROS) production (recorded using L012); this was absent when the mitochondrial flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using small interfering RNA. We propose that the Ph2 contraction is largely caused by SQR-mediated sulfide metabolism, which, by donating electrons to ubiquinone, increases electron production by complex III and thereby ROS production.

BACKGROUND AND PURPOSE:Glabridin is a major flavonoid in Glycyrrhiza glabra (licorice) root, a traditional Asian medicine. Glabridin is reported to have anti-atherogenic, anti-inflammatory and anti-nephritic properties; however its effects on vascular tone remain unexplored.EXPERIMENTAL APPROACH:We examined the effect of glabridin on rat main mesenteric artery using isometric myography and also ELISA to measure cGMP levels.KEY RESULTS:Glabridin (30μM) relaxed arteries pre-constricted with the thromboxane A2 analog U46619 (0.2μM) by ~60% in an endothelium-independent manner. Relaxation to 30μM glabridin was abolished by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (1μM) and by the BKCa channel blocker tetraethyammonium (1mM) but was unaffected by the estrogen receptor antagonist ICI182780. The concentration-response curve to glabridin (0.1 to 30μM) was downshifted by the KATP channel blocker glibenclamide (10μM), the KV channel blocker 4-aminopyridine (300μM), and the KIR blocker BaCl2 (30μM). In U46619-contracted arteries partially relaxed by 0.1μM sodium nitroprusside, application of 10 and 30nM glabridin caused additional vasorelaxation. Glabridin (30μM) approximately doubled tissue [cyclic GMP]. Application of the phosphodiesterase inhibitor isobutylmethylxanthine caused a much larger rise in [cyclic GMP], and glabridin failed to cause vasorelaxation or a further rise in [cGMP] when co-applied with IBMX.CONCLUSIONS AND IMPLICATIONS:Vasorelaxation to glabridin is dependent on the opening of K+ channels, particularly BKCa, probably caused by a rise in cellular [cyclic GMP] owing to phosphodiesterase inhibition. In the presence of sodium nitroprusside an effect of glabridin is observed at nM concentrations, similar those measured in plasma following human ingestion of licorice flavonoid oil.

Aims Sphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (<= 1 mu mol/L), however, it does not constrict intrapulmonary arteries (IPAs), but strongly potentiates vasoreactivity. Our aim was to determine whether this also occurs in a systemic artery and to delineate the signalling pathway.Methods and results Rat mesenteric arteries and IPAs mounted on amyograph were challenged with similar to 25 mu mol/L [K+] to induce a small vasoconstriction. SPC (1 mu mol/L) dramatically potentiated this constriction in all arteries by similar to 400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C (PLC; U73122), PKC epsilon (inhibitory peptide), Src (PP2), and NADPH oxidase (VAS2870), and also by Tempol (superoxide scavenger), but not by inhibition of Rho kinase (Y27632). Potentiation was lost in mesenteric arteries from p47(phox-/-), but not NOX2(-/-), mice. The intracellular superoxide generator LY83583 mimicked the effect of SPC. SPC elevated reactive oxygen species (ROS) in vascular smooth muscle cells, and this was blocked by PP2, VAS2870, and siRNA knockdown of PKC epsilon. SPC (1 mu mol/L) significantly reduced the EC50 for U46619-induced vasoconstriction, an action ablated by Tempol. In patch-clamped mesenteric artery cells, SPC (200 nmol/L) enhanced Ba2+ current through L-type Ca2+ channels, an action abolished by Tempol but mimicked by LY83583.Conclusion Our results suggest that low concentrations of SPC activate a PLC-coupled and NOX1-mediated increase in ROS, with consequent enhancement of voltage-gated Ca2+ entry and thus vasoreactivity. We speculate that this pathway is not specific for SPC, but may also contribute to vasoconstriction elicited by other G-protein coupled receptor and PLC-coupled agonists.

To determine the role of gap junctions (GJs) in hypoxic pulmonary vasoconstriction (HPV).Studies were performed in rat isolated intrapulmonary arteries (IPAs) mounted on a myograph and in anaesthetized rats. Hypoxia induced a biphasic HPV response in IPAs preconstricted with prostaglandin F-2 (PGF(2), 3 M) or 20 mM K. The GJ inhibitors 18-glycyrrhetinic acid (18-GA, 30 M), heptanol (3.5 mM), or 2-aminoethoxydiphenyl borate (2-APB) (75 M) had little effect on the transient Phase 1 of HPV, but abolished the sustained Phase 2 which is associated with Ca-2 sensitization. The voltage-dependent Ca-2 channel blocker diltiazem (10 M) had no effect on HPV, and did not alter the inhibitory action of 18-GA. Sustained HPV is enhanced by high glucose (15 mM) via potentiation of Ca-2 sensitization, in the presence of high glucose 18-GA still abolished sustained HPV. Simultaneous measurement of tension and intracellular Ca-2 using Fura PE-3 demonstrated that whilst 18-GA abolished tension development during sustained HPV, it did not affect the elevation of intracellular Ca-2. Consistent with this, 18-GA abolished hypoxia-induced phosphorylation of the Rho kinase target MYPT-1. In anaesthetized rats hypoxia caused a biphasic increase in systolic right ventricular pressure. Treatment with oral 18-GA (25 mg/kg) abolished the sustained component of the hypoxic pressor response.These results imply that GJs are critically involved in the signalling pathways leading to Rho kinase-dependent Ca-2 sensitization during sustained HPV, but not elevation of intracellular Ca-2, and may explain the dependence of the former on an intact endothelium.

Rationale: IL-13 and TGF-β have been shown to affect expression of calcium handling proteins in airway smooth muscle (ASM). We hypothesise they also alter expression of TRPC6 channel proteins. Methods: Cultured ASM cells were serum starved for 72h before treatment. SMAD2 and SMAD3 siRNAs were transfected using AmaxaTM nucleofector (Lonza). Protein expression was measured via western blots, and intracellular calcium with Fura3-PE/AM. Results: Treatment of ASM cells for 1 week with TGF-β (10ng/ml) increased TRPC6 protein (285 ± 48% n=17 p TGF-β significantly reduced SMAD3 protein (p Conclusions: TGF-β but not IL-13 results in differential increases in TRPC6 splice variant expression, regulated by SMAD2/3. As this was accompanied by reduced calcium mobilisation, we speculate increased expression of non-functional TRPC6 variants may contribute to impaired calcium handling in asthma.

Sphingosylphosphorylcholine (SPC), at concentrations that do not cause contraction (<1uM), causes potentiation of vasoconstriction to other agonists or depolarisation (Snetkov et al, Hypertension 2008;51:239–245). Our objective was to determine whether this was related to activation of NADPH oxidase and production of reactive oxygen species (ROS).METHODSSmall pulmonary or mesenteric arteries were mounted on a myograph, challenged with depolarising solutions containing ~23 mM KCl to elicit a small contraction, and incubated with SPC (1uM) before being re‐challenged.RESULTS0.2 to 1uM SPC caused substantial potentiation of depolarisation‐induced force to between 400 and 800% of control in both rat and mouse arteries. Tempol (3mM) suppressed potentiation (e.g. PA: 526 +/− 220%; +tempol: 200+/−60%, n=5). SPC enhanced ROS production estimated by lucigenin and DCF. Potentiation was abolished in p47phox −/− mice (n=6). 0.2 uM SPC enhanced voltage activated calcium current in whole cell patch by ~30% (p<0.01), and this was mimicked by exogenous ROS. We conclude that low concentrations of SPC may cause hyperreactivity via NADPH oxidase and elevation of ROS, which enhances calcium entry via L‐type channels. Funded by the Wellcome Trust

The gas H2S (sulfide) causes a biphasic contraction of unknown mechanism in pulmonary arteries (PA). We assessed this mechanism in rings of rat 2nd order PA using myography to measure tension and NaHS as a source of sulfide. Reactive oxygen species (ROS) levels and the mitochondrial membrane potential (Δψ) were recorded using the indicators LO12 and TMRE, respectively. NaHS (500μM) evoked a small contraction and then a much larger 2nd contraction which gradually relaxed. The 2nd contraction was strongly reduced by 10μg/ml antimycin (AM) and 50μM dantrolene but was unaffected by 1μM rotenone (ROT). 10 or 30μM sulfide caused an immediate transient rise in [ROS]; at 100–1000μM this was followed by a 2nd larger rise in ROS which fell to baseline within ~10 min. The biphasic increases in tension and ROS had similar timecourses. The rise in [ROS] was slightly blocked by 1μM ROT but was almost abolished by 10μg/ml AM. 500 μM sulphide caused a triphasic effect on Δψ: a rapid very transient hyperpolarization was followed by a depolarization, a 2nd slower hyperpolarization, and then a return to baseline. Sulfide oxidation by the mitochondrial flavoprotein sulfide‐quinone oxoreductase (SQR), which is expressed in PA, has been shown in some cells to generate electrons that are passed to complex 3. We propose that metabolism of sulfide by SQR gives rise to a complex 3‐mediated increase in ROS that causes a contraction by activating the RyR.

AIMS:the aim of this study was to determine the relative importance of Ca(2+) sensitization, ion channels, and intracellular Ca(2+) ([Ca(2+)](i)) in the mixed constrictor/relaxation actions of superoxide anion on systemic and pulmonary arteries.METHODS AND RESULTS:pulmonary and mesenteric arteries were obtained from rat. Superoxide was generated in arteries and cells with 6-anilino-5,8-quinolinequinone (LY83583). Following pre-constriction with U46619, 10 μmol/L LY83583 caused constriction in pulmonary and relaxation in mesenteric arteries. Both constrictor and relaxant actions of LY83583 were inhibited by superoxide dismutase and catalase. LY83583 caused Rho-kinase-dependent constriction in α-toxin-permeabilized pulmonary but not mesenteric arteries. Phosphorylation of myosin phosphatase-targeting subunit-1 (MYPT-1; as determined by western blot), was enhanced by LY83583 in pulmonary artery only. However, in both artery types, changes in tension were closely correlated with changes in phosphorylation of the 20 kDa myosin light chain as well as changes in [Ca(2+)](i) (as measured with Fura PE-3), with LY83583 causing increases in pulmonary and decreases in mesenteric arteries. When U46619 was replaced by 30 mmol/L K(+), all changes in [Ca(2+)](i) were abolished and LY83583 constricted both artery types. The K(V) channel inhibitor 4-aminopyridine abolished the LY83583-induced relaxation in mesenteric artery without affecting constriction in pulmonary artery. However, LY83583 caused a similar hyperpolarizing shift in the steady-state activation of K(V) current in isolated smooth muscle cells of both artery types.CONCLUSIONS:superoxide only causes Rho-kinase-dependent Ca(2+) sensitization in pulmonary artery, resulting in constriction, and whilst it opens K(V) channels in both artery types, this only results in relaxation in mesenteric.

J. Neurochem. (2010) 114 , 832–842. Abstract Epidemiological studies have shown an association between statin use and a decreased risk of dementia. However, the mechanism by which this beneficial effect is brought about is unclear. In the context of Alzheimer’s disease, at least three possibilities have been studied; reduction in amyloid β peptide (Aβ) production, the promotion of α‐secretase cleavage and positive effects on neurite outgrowth. By investigating the effects of mevalonate pathway blockade on neurite outgrowth using real‐time imaging, we found that rather than promote the production of neurite extensions, inhibition rapidly induced cell rounding. Crucially, neurite‐like structures were generated through the persistence of cell–cell and cell–substrate adhesions and not through a mechanism of positive outgrowth. This effect can be strikingly enhanced by the over‐expression of human amyloid precursor protein and is isoprenoid rather than cholesterol dependent.

Neural induction is the first step in the formation of the vertebrate central nervous system. The emerging consensus of the mechanisms underling neural induction is the combined influences from inhibiting bone morphogenetic protein (BMP) signaling and activating fibroblast growth factor (FGF)/Erk signaling, which act extrinsically via either autocrine or paracrine fashions. However, do intrinsic forces (cues) exist and do they play decisive roles in neural induction? These questions remain to be answered. Here, we have identified a novel neural initiator, neuronatin (Nnat), which acts as an intrinsic factor to promote neural fate in mammals and Xenopus. ESCs lacking this intrinsic factor fail to undergo neural induction despite the inhibition of the BMP pathway. We show that Nnat initiates neural induction in ESCs through increasing intracellular Ca2+ ([Ca2+]i) by antagonizing Ca2+‐ATPase isoform 2 (sarco/endoplasmic reticulum Ca2+‐ATPase isoform 2) in the endoplasmic reticulum, which in turn increases the phosphorylation of Erk1/2 and inhibits the BMP4 pathway and leads to neural induction in conjunction with FGF/Erk pathway. STEM CELLS 2010;28:1950–1960

The src family of tyrosine kinases (srcFK) are important upstream regulators of Rho‐kinase mediated Ca2+‐sensitization and constriction in rat pulmonary artery (RPA). We hypothesise that srcFK act through modulation of the activity of RhoA. Cytosolic to membrane translocation of RhoA in RPA smooth muscle cells, visualised using over‐expressed green fluorescent protein‐tagged RhoA was used as an indication of RhoA activation. Translocation was stimulated by prostaglandin‐F2α (20 μM), the superoxide generating quinone LY83583 (10 μM), and the RhoA activator calpeptin (0.02 U/ml). This translocation, following all three types of stimulus, was prevented by prior incubation with the srcFK antagonist PP2 (10 μM). Calpeptin also constricted myograph‐mounted RPA, and this constriction was inhibited by PP2. srcFK are likely to be activating RhoA through phosphorylation of one or more of the many proteins that directly activate or inhibit RhoA, namely guanine nucleotide exchange factors (RhoGEF), GTPase activating proteins (RhoGAP), and guanine nucleotide dissociation inhibitors (RhoGDI). We used RT‐PCR to confirm endogenous expression of p115‐RhoGEF, PDZ‐RhoGEF, LARG, Vsm‐RhoGEF, SmgGDS, vav‐3, p73‐RhoGAP, RhoGDI‐1 and RhoGDI‐2 in RPA. The GEF Vav‐2 was not expressed. Our data suggests that srcFK may be contributing to Ca2+‐sensitization and constriction of RPA through activation of RhoA.