Untreated Wild-type Research Articles

Extra domain A-containing fibronectin in pulmonary hypertension and treatment effects of a function-blocking antibody.

Pulmonary vascular and right ventricular (RV) remodelling processes are important for development and progression of pulmonary hypertension (PH). The current study analysed the functional role of the extra domain A-containing fibronectin (ED-A+ Fn) for the development of PH by comparing ED-A+ Fn knockout (KO) and wild-type (WT) mice as well as the effects of an antibody-based therapeutic approach in a model of monocrotaline (MCT)-induced PH, which will be validated in a model of Sugen 5416/hypoxia-induced PH. PH was induced using MCT (PH mice). Sixty-nine mice were divided into the following groups: sham-treated controls (WT: n = 7; KO: n = 7), PH mice without specific treatment (WT: n = 12; KO: n = 10), PH mice treated with a dual endothelin receptor antagonist (macitentan; WT: n = 6; KO: n = 11), WT PH mice treated with the F8 antibody, specifically recognizing ED-A+ Fn, (n = 8), and WT PH mice treated with an antibody of irrelevant antigen specificity (KSF, n = 8). Compared to controls, WT_PH mice showed a significant elevation of the RV systolic pressure (P = 0.04) and RV functional impairment including increased basal RV (P = 0.016) diameter or tricuspid annular plane systolic excursion (P = 0.008). In contrast, KO PH did not show such effects compared to controls (P = n.s.). In WT_PH mice treated with F8, haemodynamic and echocardiographic parameters were significantly improved compared to untreated WT_PH mice or those treated with the KSF antibody (P < 0.05). On the microscopic level, KO_PH mice showed significantly less tissue damage compared to the WT_PH mice (P = 0.008). Furthermore, lung tissue damage could significantly be reduced after F8 treatment (P = 0.04). Additionally, these findings could be verified in the Sugen 5416/hypoxia mouse model, in which F8 significantly improved echocardiographic, haemodynamic, and histologic parameters. ED-A+ Fn is of crucial importance for PH pathogenesis representing a promising therapeutic target in PH. We here show a novel therapeutic approach using antibody-mediated functional blockade of ED-A+ Fn capable of attenuating and partially reversing PH-associated tissue remodelling.

Mutual antagonism between CD44 and integrins in glioblastoma cell traction and migration.

Cell migration is the major driver of invasion and metastasis during cancer progression. For cells to migrate, they utilize the actin-myosin cytoskeleton and adhesion molecules, such as integrins and CD44, to generate traction forces in their environment. CD44 primarily binds to hyaluronic acid (HA) and integrins primarily bind to extracellular matrix (ECM) proteins such as collagen. However, the role of CD44 under integrin-mediated conditions and vice versa is not well known. Here, we performed traction force microscopy (TFM) on U251 cells seeded on collagen I-coated polyacrylamide gels to assess the functional mechanical relationship between integrins and CD44. Performing TFM on integrin-mediated adhesion conditions, i.e., collagen, we found that CD44KO U251 cells exerted more traction force than wild-type (WT) U251 cells. Furthermore, untreated WT and CD44-blocked WT exhibited comparable results. Conversely, in CD44-mediated adhesive conditions, integrin-blocked WT cells exerted a higher traction force than untreated WT cells. Our data suggest that CD44 and integrins have a mutually antagonistic relationship where one receptor represses the other's ability to generate traction force on its cognate substrate.

Cyclopropanation and membrane unsaturation improve antibiotic resistance of swarmer Pseudomonas and its sod mutants exposed to radiations, in vitro and in silico approch.

It was known that UVc irradiation increases the reactive oxygen species' (ROS) levels in bacteria hence the intervention of antioxidant enzymes and causes alsochanges in fatty acids (FAs) composition enabling bacteria to face antibiotics. Here, we intended to elucidate an interrelationship between SOD and susceptibility to antibiotics by studying FA membrane composition of UVc-treatedP. aeruginosaPAO1 and its isogenic mutants (sodM,sodBandsod MB) membrane, after treatment with antibiotics. Swarmer mutants defective in genes encoding superoxide dismutase were pre-exposed to UVc radiations and then tested by disk diffusion method for their contribution to antibiotic tolerance in comparison with the P. aeruginosa wild type (WT). Moreover, fatty acid composition of untreated and UVc-treated WT and sod mutants was examined by Gaz chromatography and correlated to antibiotic resistance. Firstly, it has been demonstrated that after UVc exposure, swarmer WT strain,sodMandsodBmutantsremain resistant to polymixin B, a membrane target antibiotic, through membrane unsaturation supported by the intervention of Mn-SOD after short UVc exposure and cyclopropanation of unsaturated FAs supported by the action of Fe-SOD after longer UVc exposure. However, resistance for ciprofloxacin is correlated with increase in saturated FAs. This correlation has been confirmed by a molecular docking approach showing that biotin carboxylase, involved in the initial stage of FA biosynthesis, exhibits a high affinity for ciprofloxacin. This investigation has explored the correlation of antibiotic resistance with FA content of swarmerP.aeruginosapre-exposed to UVc radiations, confirmed to be antibiotic target dependant.

AG-946, an Activator of Pyruvate Kinase, Improves Ineffective Erythropoiesis in the Bone Marrow of Mouse Models of Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematologic malignancies characterized by ineffective erythropoiesis. Anemia is the most common cytopenia and major clinical problem in MDS. Acquired pyruvate kinase (PK) deficiency has been observed in MDS, and a direct impact on the glycolytic pathway in the pathogenesis of anemia associated with MDS has been demonstrated. AG-946 is an investigational, small-molecule, allosteric activator of PK that has the potential to enhance red blood cell (RBC) functionality and survival by increasing glycolysis and adenosine triphosphate (ATP) production, and improve differentiation of erythroid cells in bone marrow, potentially improving anemia caused by ineffective erythropoiesis in MDS. Previous findings showed that RBCs from patients with MDS treated ex-vivo with AG-946, led to an increase in PK activity. Here, we used two MDS-related mouse models to evaluate the effects of the PK activator AG-946 in this mechanism of anemia. Aim: Evaluate the effect of AG-946 treatment on impaired erythropoiesis in the bone marrow of two mouse models of MDS. Methods:NUP98-HOXD13 (NHD13) is a fusion gene mutation observed in some patients with MDS. Mouse models with this mutation develop key characteristics of MDS such as impaired erythropoiesis and progressive anemia leading to malignant leukemia around 12 months of age. Male and female NHD13 and C57BL/6J mice were placed on 0.006% AG-946 (approximate dose 10 mg/kg/day) formulated diet starting at 10 months given ad libitum and continuing for 3 months. Numbers of animals for wild type (WT) Control, WT AG-946, NHD13 Control, NHD13 AG-946 were 10, 10, 13, and 11, respectively.Polg D257A (Polg) mouse model, while not a mutation observed in patients with MDS, does display many key features of MDS, such as impaired erythropoiesis and progressive anemia. Male and female Polg mice were placed on 0.006% AG-946 (approximate dose 10 mg/kg/day) formulated diet starting at 4 months given ad libitum and continuing for 8 months. Numbers of animals for WT Control, WT AG-946, Polg Control, Polg AG-946 were 10, 10, 19, and 18, respectively. Bone marrow from 1 femur per mouse was collected and analyzed by flow cytometry. Bone marrow cell pellets were washed and treated with ACK lysing buffer prior to staining. Samples with fewer than 1000 Ter119+ events were removed from analysis due to insufficient cell count. Bone marrow erythroblast populations were gated using Single Cells/Live/B220-/Ter119+/CD44 vs FSC, which were then gated into three populations: basophilic erythroblasts (BasoE), polychromatic erythroblasts (PolyE), and orthochromatic erythroblasts and reticulocytes (OrthoE + Retic). Results:No differences in any erythroblast populations were observed between untreated WT and NHD13 mice. No changes in the BasoE population were observed in WT or NHD13 mice. The PolyE population in NHD13 mice treated with AG-946 was significantly decreased compared with both the NHD13 and WT untreated mice (p=0.0021 and 0.0003, respectively). NHD13 mice treated with AG-946 showed significant increases in the OrthoE + Retic populations over the untreated NHD13 mice (p=0.0275).No differences in BasoE or OrthoE + Retic populations were observed between untreated WT and Polg mice while the PolyE population was significantly decreased in Polg mice compared with WT (p&amp;lt;0.0001). The BasoE population was significantly decreased in Polg mice treated with AG-946 compared with untreated Polg (p=0.0002). No difference in PolyE population was observed in Polg mice with AG-946. OrthoE + Retic population was significantly increased in Polg mice treated with AG-946 compared with WT and Polg untreated mice (p=0.0001 and 0.0304, respectively). Data graphed as average +/- SEM. Conclusions: Our data suggest AG-946 treatment improves erythroblast maturation in two MDS mouse models as demonstrated by a reduction in early-stage erythroblasts (BasoE, PolyE) coupled with an increase in late-stage erythroblast populations (OrthoE + Retic). These data are the first to suggest that PK activation by AG-946 could improve ineffective erythropoiesis in MDS.

UV mutagenesis followed by hydrogen peroxide treatment ameliorates lipid production and omega-3 fatty acids levels in Chlorella sp.

The high potential Chlorella sp. was obtained after the UV mutation which produces higher biomass and lipid content than the wild type. The lipid content and biomass were increased from 34 to 48 % dry cell weight and 1.42 to 1.97 g/L respectively. Further, hydrogen peroxide (H2O2) treatment increased both biomass and lipid content in mutant, whereas wild type did not show any considerable change in both biomass and lipid content. A 3-fold increase in total lipid yield was observed after the H2O2 treatment in mutant when compared to the untreated wild type. H2O2 treatment also altered the fatty acid composition of lipids resulting in enhanced eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content. Overall results indicate that the strain improvement of Chlorella sp. by UV mutagenesis and oxidative stress by H2O2 treatment enhances the biomass and lipid content with high amounts of omega-3 fatty acids. Furthermore, the H2O2 treated mutant cells showed upregulation of the important genes involved in the fatty acid synthesis pathway.

Early postnatal microglial ablation in the Ccdc39 mouse model reveals adverse effects on brain development and in neonatal hydrocephalus

BackgroundNeonatal hydrocephalus is a congenital abnormality resulting in an inflammatory response and microglial cell activation both clinically and in animal models. Previously, we reported a mutation in a motile cilia gene, Ccdc39 that develops neonatal progressive hydrocephalus (prh) with inflammatory microglia. We discovered significantly increased amoeboid-shaped activated microglia in periventricular white matter edema, reduced mature homeostatic microglia in grey matter, and reduced myelination in the prh model. Recently, the role of microglia in animal models of adult brain disorders was examined using cell type-specific ablation by colony-stimulating factor-1 receptor (CSF1R) inhibitor, however, little information exists regarding the role of microglia in neonatal brain disorders such as hydrocephalus. Therefore, we aim to see if ablating pro-inflammatory microglia, and thus suppressing the inflammatory response, in a neonatal hydrocephalic mouse line could have beneficial effects.MethodsIn this study, Plexxikon 5622 (PLX5622), a CSF1R inhibitor, was subcutaneously administered to wild-type (WT) and prh mutant mice daily from postnatal day (P) 3 to P7. MRI-estimated brain volume was compared with untreated WT and prh mutants P7-9 and immunohistochemistry of the brain sections was performed at P8 and P18-21.ResultsPLX5622 injections successfully ablated IBA1-positive microglia in both the WT and prh mutants at P8. Of the microglia that are resistant to PLX5622 treatment, there was a higher percentage of amoeboid-shaped microglia, identified by morphology with retracted processes. In PLX-treated prh mutants, there was increased ventriculomegaly and no change in the total brain volume was observed. Also, the PLX5622 treatment significantly reduced myelination in WT mice at P8, although this was recovered after full microglia repopulation by P20. Microglia repopulation in the mutants worsened hypomyelination at P20.ConclusionsMicroglia ablation in the neonatal hydrocephalic brain does not improve white matter edema, and actually worsens ventricular enlargement and hypomyelination, suggesting critical functions of homeostatic ramified microglia to better improve brain development with neonatal hydrocephalus. Future studies with detailed examination of microglial development and status may provide a clarification of the need for microglia in neonatal brain development.

NON-PHARMACOLOGICAL INTERVENTIONS UP-REGULATE MYOCARDIAL CONNEXIN-43 AND DECREASE A RISK OF MALIGNANT ARRHYTHMIAS IN EXPERIMENTAL MODEL OF ESSENTIAL HYPERTENSION

Objective: Available data suggest that meta-inflammation and oxidative stress as well as endogenous deficiency of omega-3 and melatonin may be involved in hypertension-induced myocardial abnormalities that increase propensity to cardiac arrhythmias. We aimed to explore whether supplementation with omega-3 and melatonin as well as cold acclimation may affect arrhythmogenicity of rats suffering from essential hypertension. We focused on myocardial connexin-43(Cx43) channel protein, which ensures cardiac cell-cell coupling for electrical signal propagation, thereby is crucial factor impacting susceptibility of the heart malignant arrhythmias. Design and method: Experiments were performed using adult, male, spontaneously hypertensive rats (SHR, wild type), hairless SHR (mutant strain) and normotensive Wistar rats. SHR were treated for two-month with omega-3 (Omacor,200mg/day) or melatonin (400ug/day) and compared with untreated rats. Hairless SHR adapted to cold were compared with wild type SHR and normotensive Wistar rats. Biometrical parameters were registered and left ventricular heart tissue was taken for analysis of Cx43 and PKC↙, which via phosphorylation affects Cx43 channel function. Isolated perfused heart was used for the examination of its susceptibility to electrically-induced sustained ventricular fibrillation (VF). Results: Supplementation with omega-3 or melatonin and cold acclimation did not affect significantly blood pressure as well as heart or left ventricular weights of SHR or hairless SHR. However, the electrical threshold to induce VF was significantly lower (∼20mA) in non-treated compared with treated SHR (∼35mA) as well as in wild type SHR compared to hairless SHR (∼40mA). Antiarrhythmic effect was associated with increased protein levels of Cx43 and its functional phosphorylated forms in left ventricles of treated SHR and hairless SHR compared to untreated wild type SHR. Moreover, pro-arrhythmic myocardial Cx43 distribution on lateral sides of the cardiomyocytes was attenuated by treatment or cold acclimation. In parallel, there was an increase of PKC↙ expression that may enhance cardio-protective Cx43 phosphorylation. Conclusions: In conclusion, findings indicate that non-pharmacological interventions can protect hypertensive rat heart from malignant arrhythmias, at least in part, by targeting myocardial Cx43. It is challenging to pay attention to non-pharmacological approaches in humans suffering from primary hypertension.

ABCA3 Deficiency-Variant-Specific Response to Hydroxychloroquine.

Biallelic variants in ABCA3, the gene encoding the lipid transporter ATP-binding cassette subfamily A member 3 (ABCA3) that is predominantly expressed in alveolar type II cells, may cause interstitial lung diseases in children (chILD) and adults. Currently, there is no proven therapy, but, frequently, hydroxychloroquine (HCQ) is used empirically. We hypothesized that the in vitro responsiveness to HCQ might correlate to patients' clinical outcomes from receiving HCQ therapy. The clinical data of the subjects with chILD due to ABCA3 deficiency and treated with HCQ were retrieved from the literature and the Kids Lung Register data base. The in vitro experiments were conducted on wild type (WT) and 16 mutant ABCA3-HA-transfected A549 cells. The responses of the functional read out were assessed as the extent of deviation from the untreated WT. With HCQ treatment, 19 patients had improved or unchanged respiratory conditions, and 20 had respiratory deteriorations, 5 of whom transiently improved then deteriorated. The in vitro ABCA3 functional assays identified two variants with complete response, five with partial response, and nine with no response to HCQ. The variant-specific HCQ effects in vivo closely correlated to the in vitro data. An ABCA3+ vesicle volume above 60% of the WT volume was linked to responsiveness to HCQ; the HCQ treatment response was concentration dependent and differed for variants in vitro. We generated evidence for an ABCA3 variant-dependent impact of the HCQ in vitro. This may also apply for HCQ treatment in vivo, as supported by the retrospective and uncontrolled data from the treatment of chILD due to ABCA3 deficiency.

Lipidomic landscape of circulating extracellular vesicles isolated from adolescents exposed to ethanol intoxication: a sex difference study

BackgroundLipids represent essential components of extracellular vesicles (EVs), playing structural and regulatory functions during EV biogenesis, release, targeting, and cell uptake. Importantly, lipidic dysregulation has been linked to several disorders, including metabolic syndrome, inflammation, and neurological dysfunction. Our recent results demonstrated the involvement of plasma EV microRNAs as possible amplifiers and biomarkers of neuroinflammation and brain damage induced by ethanol intoxication during adolescence. Considering the possible role of plasma EV lipids as regulatory molecules and biomarkers, we evaluated how acute ethanol intoxication differentially affected the lipid composition of plasma EVs in male and female adolescents and explored the participation of the immune response.MethodsPlasma EVs were extracted from humans and wild-type (WT) and Toll-like receptor 4 deficient (TLR4-KO) mice. Preprocessing and exploratory analyses were conducted after the extraction of EV lipids and data acquisition by mass spectrometry. Comparisons between ethanol-intoxicated and control human female and male individuals and ethanol-treated and untreated WT and TLR4-KO female and male mice were used to analyze the differential abundance of lipids. Annotation of lipids into their corresponding classes and a lipid set enrichment analysis were carried out to evaluate biological functions.ResultsWe demonstrated, for the first time, that acute ethanol intoxication induced a higher enrichment of distinct plasma EV lipid species in human female adolescents than in males. We observed a higher content of the PA, LPC, unsaturated FA, and FAHFA lipid classes in females, whereas males showed enrichment in PI. These lipid classes participate in the formation, release, and uptake of EVs and the activation of the immune response. Moreover, we observed changes in EV lipid composition between ethanol-treated WT and TLR4-KO mice (e.g., enrichment of glycerophosphoinositols in ethanol-treated WT males), and the sex-based differences in lipid abundance are more notable in WT mice than in TLR4-KO mice. All data and results generated have been made openly available on a web-based platform (http://bioinfo.cipf.es/sal).ConclusionsOur results suggest that binge ethanol drinking in human female adolescents leads to a higher content of plasma EV lipid species associated with EV biogenesis and the propagation of neuroinflammatory responses than in males. In addition, we discovered greater differences in lipid abundance between sexes in WT mice compared to TLR4-KO mice. Our findings also support the potential use of EV-enriched lipids as biomarkers of ethanol-induced neuroinflammation during adolescence.

PS-B01-12: SEXUAL DIMORPHISM OF CEREBROVASCULAR NOX5-NOTCH3 SIGNALLING IN ANGIOTENSIN II-INDUCED HYPERTENSION

Objectives: NOX5 is a pro-contractile NOX isoform associated with vascular dysfunction. We demonstrated that vascular NOX5 expression is increased in cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy (CADASIL), a small vessel disease of the brain. CADASIL is caused by mutations in NOTCH3 and abnormal NOTCH3 signalling. Here we questioned whether cerebrovascular NOX5 influences NOTCH3 in male and female hypertensive mice. Design and method: Brain tissues from angiotensin II (Ang II) male and female 20-week wild-type (WT) ice and mice expressing human Nox5 (hNOX5) specifically in VSMCs studied. Ang II was administered by mini-pump (600ng/Kg/per day) for 28 days to induce a hypertension. Protein and mRNA expression of NOX isoforms and NOTCH3 (full-length and intracellular domain (ICD)) and downstream signalling of NOTCH3-specific genes were assessed using qPCR and immunoblotting respectively. Markers of oxidative stress were measured by Amplex-red and TBARS-MDA. Results: Ang II induced a significant increase in blood pressure in WT and hNOX5 mice. Expression of full length NOTCH3 expression was significantly increased in male Ang II-treated hNOX5 mice (AU: 0.15 ± 0.01 vs. un-treated hNOX5 0.07 ± 0.02) and unchanged in females. NOTCH3-ICD was significantly increased in male Ang II-treated WT versus WT controls (0.1 ± 0.01 untreated WT vs. 0.25 ± 0.05 Ang II WT), but unchanged in male hNOX5 mice. NOTCH3-ICD expression was unaltered in female untreated versus Ang II treated WT and hNOX5 mice. Downstream NOTCH3 gene HEY1 mRNA expression was significantly reduced in male Ang II-treated hNOX5 mice compared to untreated hNOX5 males (3.91 ± 0.3 vs. male hNOX5 untreated 2.78 ± 0.13). Irreversible protein tyrosine phosphatases (PTP-ox) were significantly increased in Ang II-treated hNOX5 mice (AU: 4.91 ± 0.6 vs. 2.18 ± 0.14) and was unchanged in hNOX5 female mice. MDA lipid peroxidation markers were decreased in female hNOX5 Ang II-treated mice (umol/protein: 1.28 ± 0.26 vs. hNOX5 3.25 ± 0.43), but not males. NOX5 induced no changes in NOX1-4 protein expression, or ROS measures in males or females. Conclusions: Ang II-induced hypertension in VSMC-specific NOX5 mice leads abnormal NOTCH3 signalling, downstream HEY1 transcription, and increased PTP-ox in a sex-dependent manner. In conclusion, in brains from VSMC-specific hNOX5 mice under a hypertensive phenotype, NOTCH3 expression is increased with associated attenuation of NOTCH3 downstream gene expression and increased PTP signalling. These findings were only evident in male mice. Our data suggest a possible relationship between NOTCH3 signalling and NOX5 that seems to be sex-specific. Implications of this sexual dimorphism await further clarification in the context of hypertension and vascular dysfunction.

Neuronal melatonin type 1 receptor overexpression promotes M2 microglia polarization in cerebral ischemia/reperfusion-induced injury

Microglial activation is readily detected following cerebral ischemia/reperfusion-induced injury. Activated microglia polarize into either classic pro-inflammatory M1 or protective M2 microglia following ischemia/reperfusion-induced injury. Melatonin is protective immediately after ischemia/reperfusion-induced brain injury. However, the ability of melatonin to affect longer-term recovery from ischemic/reperfusion-induced injury as well as its ability to modulate microglia/macrophage polarization are unknown. The goal of this study is to understand the impact of melatonin on mice 14 days after injury, as well as to understand how melatonin affects microglial polarization of neuronal MT1 activation following cerebral ischemia/reperfusion. We utilized NSEMT1-GFP transgenic mice which overexpress MT1 (melatonin type 1 receptor) in neurons. Melatonin-treated or vehicle treated wild type and NSEMT1-GFP mice underwent middle cerebral artery occlusion (MCAO)/reperfusion and followed for 14 days. Neuronal MT1 overexpression significantly reduced infarct volumes, improved motor function, and ameliorated weight loss. Additionally, melatonin treatment reduced infarct volume in NSEMT1-GFP mice as compared to untreated wild type, melatonin treated wild type, and untreated NSEMT1-GFP mice. Melatonin improved neurological function and prevented weight loss in NSEMT1-GFP mice compared with melatonin treated wild type mice. Finally, melatonin treatment in combination with MT1 overexpression reduced the numbers of Iba1+/CD16+ M1 microglia and increased the numbers of Iba1+/ CD206+ M2 microglia after ischemic injury. In conclusion, neuronal MT1 mediates melatonin-induced long-term recovery after cerebral ischemia, at least in part, by shifting microglial polarization toward the neuroprotective M2 phenotype.

COP1 mutation causes low leaf temperature under various abiotic stresses in Arabidopsis thaliana.

Stomata are microscopic pores on epidermal cells of leaves and stems that regulate water loss and gas exchange between the plant and its environment. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase that is involved in plant growth and development and multiple abiotic stress responses by regulating the stability of various target proteins. However, little is known about how COP1 controls stomatal aperture and leaf temperature under various environmental conditions. Here, we show that COP1 participates in leaf temperature and stomatal closure regulation under normal and stress conditions in Arabidopsis. Leaf temperature of cop1 mutants was relatively lower than that of wild type (WT) under drought, salt, and heat stress and after abscisic acid (ABA), CaCl2, and H2O2 treatments. However, leaf temperature was generally higher in both WT and cop1 mutants after abiotic stress and chemical treatment than that of untreated WT and cop1 mutants. Stomatal aperture was wider in cop1 mutants than that in WT under all conditions tested, although the extent of stomatal closure varied between WT and cop1 mutants. Under dark conditions, leaf temperature was also lower in cop1 mutants than that in WT. Expression of the genes encoding ABA receptors, ABA biosynthesis proteins, positive regulators of stomatal closure and heat tolerance, and ABA-responsive proteins was lower in cop1 mutants that that in WT. In addition, expression of respiration-related genes was lower in cop1 mutants that that in WT. Taken together, the data provide evidence that mutations in COP1 lead to wider stomatal aperture and higher leaf temperature under normal and stress conditions, indicating that leaf temperature is highly correlated with stomatal aperture.

Metformin Treatment Abrogates the Competitive Advantage of Dnmt3a R878H HSPCs By Enhancing DNA Methylation Activity

Clonal hematopoiesis of indeterminate potential (CHIP) refers to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) carrying leukemogenic mutations in individuals without evidence of a blood disorder. CHIP carriers are at an increased risk of blood cancers and cardiovascular diseases. Targeting the clonal expansion of mutant HSPCs has the potential to mitigate CHIP-related illnesses. The most commonly mutated gene in CHIP is DNMT3A, which encodes an enzyme that catalyzes the methyl transfer from S-adenosylmethionine (SAM) to cytosines in DNA. The heterozygous DNMT3A hotspot mutation at R882 exerts a dominant negative effect on the wild-type (WT) allele, resulting in decreased DNA methylation activity. Prior studies have shown that murine HSPCs carrying mutant Dnmt3a R878H (equivalent to R882 in humans) have an advantage over WT cells in competition assays. Here, we aimed to first identify differences in the functional properties between Dnmt3a-mutated and WT HSPCs. Given the crosstalk between mitochondrial metabolism and DNA methylation, we investigated potential differences in mitochondrial activity and found that the level of mitochondrial respiration was higher in Dnmt3aR878H/+ HSPCs than in WT cells. To determine if this translated to differential dependencies, we silenced the expression of critical subunits in the each of five electron transport chain (ETC) complexes. Knockdown of NDUFV1 in Complex I was the most effective in reducing the competitive advantage of Dnmt3aR878H/+ cells in vitro. To explore translational relevance of this finding, we tested the impact of metformin, a pharmacologic inhibitor of Complex I, on Dnmt3a-mutated HSPCs. Treatment with metformin at clinically relevant concentrations suppressed the competitive advantage of mutant cells in vitro. Importantly, this effect was rescued by expression of NDI1, a metformin-resistant yeast analog of Complex I. To extend these findings, we conducted a competitive repopulation assay between CD45.2+ Dnmt3aR878H/+ and CD45.1+ Dnmt3a+/+ HSPCs. Metformin treatment abrogated the in vivo competitive advantage of mutant cells over an 8-month period based on peripheral blood chimerism. To decipher the mechanism of action, we performed transcriptome and metabolome profiling of mutant HSPCs treated with or without metformin for 1 month. GSEA analysis of the transcriptome data revealed a downregulation of genes associated with stemness in metformin-treated cells. Intriguingly, we found that metformin increased the expression of genes involved in one-carbon metabolism, which generates SAM. Consistent with these findings, metabolomics analysis showed that the levels of several key metabolites in one-carbon metabolism, including SAM, were higher in metformin-treated cells compared with controls. Genetic and pharmacologic inhibition of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism, as well as the addition of exogeneous S-adenosylhomocysteine (SAH), an inhibitor of SAM-dependent methyltransferases, effectively rescued the effect of metformin on mutant cells in vitro. Based on the above findings, we hypothesized that metformin treatment might enhance the residual DNA methylation activity found in Dnmt3aR878H/+ mutated cells by increasing SAM concentration. In support of this hypothesis, we found that exogeneous SAM reproduced the effect of metformin, and HSPCs with homozygous Dnmt3aR878H mutations were insensitive to metformin. Furthermore, we performed DNA methylome profiling of untreated and metformin-treated mutant and WT HSPCs using RRBS. This analysis revealed that the hypomethylated regions (HMRs) in untreated Dnmt3aR878H/+ HSPCs relative to WT controls were enriched for regions of repressed chromatin with H3K27 methylation. Importantly, metformin treatment increased the level of DNA methylation at these regions in mutated HSPCs. In summary, our findings demonstrate that Dnmt3aR878H/+ HSPCs depend on ETC activity and particularly complex I activity to maintain their competitive advantage over WT cells. We propose a model in which inhibition of complex I activity with metformin increases one-carbon metabolism and SAM level, thereby restoring DNA methylation at HMRs and the differentiation of mutant HSPCs. Our findings have important implications for the development of preventive interventions against DNMT3A-mutated CHIP.

Revisiting the anticancer properties of phosphane(9-ribosylpurine-6-thiolato)gold(I) complexes and their 9H-purine precursors

New mono- and di-nuclear thio-purine and thio-purine nucleoside gold(I) complexes were synthesized, characterized, and evaluated in vitro for biological activities in comparison to related known purine complexes. By combining known anti-tumoral thio-purines with R3PAu moieties as present in auranofin, complexes with enhanced effects and selectivities were obtained, which not only act as cytostatics, but also disrupt tumor-specific processes. Their IC50 values in cytotoxicity test with tumor cell lines ranged from three-digit nanomolar to single-digit micromolar, revealing a tentative structure–activity relationship (SAR). Both the residues R2 of the phosphane ligand and R1 at C2 of the pyrimidine ring had a significant impact on the cytotoxicity. In most cases, the introduction of a ribo-furanosyl group at N9 of the purine led to a distinctly more cytotoxic complex. Most complexes were more active against multi-drug-resistant tumor cells or such lacking functional p53 when compared to the respective untreated wild type cell lines. Some nucleoside complexes displayed an interesting dose-dependent dual mode of action regarding cell cycle arrest and DNA repair mechanism. Some phosphane(purine-6-thiolato)gold (I) complexes had a stronger inhibitory effect on the thioredoxin reductase (TrxR) and on the reactive oxygen species (ROS) generation in cancer cells than is typical of other gold complexes. They also led to DNA fragmentation and showed anti-angiogenic effects. Their stability under test conditions was demonstrated by 77Se NMR monitoring of an exemplary selenopurine complex.Graphical abstract

PEOPLE (NTC03447678), a phase II trial of first-line, single-agent pembrolizumab in advanced NSCLC with low PD-L1: Clinical outcomes and association with circulating immune biomarkers.

9033 Background: In advanced NSCLC (aNSCLC) with PD-L1 &lt; 50% chemo-immunotherapy is the standard of care. Although the activity of single agent pembrolizumab was reported, no biomarkers have been identified able to select patients who mostly benefit. The trial aimed to identify immune biomarkers associated with PFS in aNSCLC patients with PD-L1 levels &lt; 50% treated with first-line pembrolizumab. Here we reported for the first time the clinical outcomes of the trial and circulating immune profiling (CIP) biomarkers correlated to PFS. Methods: This phase II trial was conducted at Fondazione IRCCS Istituto Nazionale dei Tumori of Milan. Eligible patients were previously untreated stage IIIB-IV NSCLC, EGFR and ALK wild type with PD-L1 &lt; 50%, PS 0-2. PD-L1 was assessed with 22-C3 antibody (Dako). Patients received pembrolizumab 200 mg every 3 weeks until 35 cycles, disease progression or unacceptable toxicity. The primary endpoint was the association of immune biomarkers with PFS. OS, ORR, DCR, DoR and safety were secondary endpoints. CIP was performed by determination of absolute cell counts for 36 innate and adaptive immunity subsets through multiparametric flow cytometry on freshly isolated whole blood samples at baseline. An orthoblique principal components-based clustering approach and multivariable Cox regression model adjusted for clinical variables were used to analyse CIP variables and their correlation with clinical endpoints. Results: From May 2018 to October 2020, of 87 screened, 65 patients were enrolled. The median age was 70.9 years, most patients were male (67.7%), smoker (87.7%), non-squamous (83.1%), PDL1 + (70.8%). 12 patients (18.5%) had PS 2. During a median follow-up of 26.4 months (mo), 51 radiological progressions, 46 deaths and 60 PFS events were observed. The median PFS was 2.9 mo (95%CI 1.8 - 5.6) and the median OS was 12.1 mo (95%CI 8.7 - 17.1). The ORR was 24.1% (2 complete and 12 partial responses), DCR was 53.4% and median DoR was 14.5 mo (95%CI 6.4 - 24.9). Drug related G3-G4 adverse events were: 2 hyponatremia, 2 lipase increased, 1 pneumonitis. Out of 7 CIP clusters identified, 2 were statistically significant at multivariable analysis on 57 patients. Higher baseline counts of 8 subsets within these two clusters were associated with better PFS (HR values range: 0.88-0.98; p values range: 0.001- 0.016). The significant subsets included lymphocytes (cells expressing CD3, or CD19 or CD56, but lacking granulocyte and monocyte markers) and main NK subsets (including CD56dim CD16+, CD56dim CD16- and HLA-DR+ NK cells). Conclusions: This trial confirmed that pembrolizumab as first-line single agent is safe and active also in a subgroup of aNSCLC patients with PD-L1 &lt; 50%. CIP biomarkers can be useful to identify patients with a favourable outcome, thus avoiding the adding toxicity of chemotherapy. Clinical trial information: NTC03447678.

Dysfunctional Mitochondria and Hemolysis are Linked by Impaired Maturation of Reticulocytes: Possible Role in Pulmonary Hypertension

IntroductionWe previously demonstrated that significantly elevated levels of circulating free heme (CFH) is associated with preclinical (Sugen/Hypoxia in rats) pulmonary hypertension (PH) and clinical pulmonary arterial hypertension (PAH). However, the pathogenic mechanisms of free heme release in PAH patients and animal models in the absence of hemolytic disease are yet to be elucidated.HypothesisThe mitochondria play a crucial role in reticulocyte maturation (precursors to red blood cells). Our hypothesis tests if mitochondrial dysfunction (MD) impaired reticulocyte maturation increases its fragility and causes rupture, elevating CFH levels that promote PAH.MethodsRecently reported genetic PH rat models carrying the NFU1G206C mutation spontaneously develop PH with severe MD. Erythropoiesis activation, reticulocyte counts, and CFH levels were monitored in this model. ChemiDoc imaging system was used to visualize FITC‐dextran extravasation in fresh transversely cut pulmonary slices 24 hours after FITC‐dextran injection (60 mg/kg. i.v.). Changes in right ventricle (RV) hemodynamics were used to determine the level of disease severity. Pulmonary artery smooth muscle cells (PASMCs) isolated from wild‐type (WT) and NFU1G206C rats were used to evaluate effects of free heme in vitro. Matrigel assay was used to evaluate the tube formation potential of endothelial cells (EC’s).ResultsThe NFU1G206C rats showed significantly higher erythropoietin levels and immature reticulocyte counts compared to WTs. Correspondingly, significantly elevated plasma free heme and extravasation of FITC‐dextran into lung parenchyma were observed, indicating pulmonary endothelial barrier dysfunction, previously demonstrated as an essential contributor to PH pathobiology. Notably, treatment of human pulmonary artery EC’s with 50µM heme completely abolished EC tube formation, indicating that CFH could trigger a cellular environment leading to PH. Additionally, exposing isolated PASMC from NFU1G206C to 50µM heme increased the proliferation rate by ~1.5 and ~2.9 folds compared to untreated NFU1G206C or WT PASMC. Chronic lipoic acid (LA) supplementation to restore mitochondrial function (1.7mM orally), normalized total/immature reticulocyte counts in NFU1G206C rats (WT/NFU1G206C/NFU1G206C+LA for total and immature reticulocyte, p&lt;0.002 and p&lt;0.001) and fully reversed the PAH phenotype. Alternatively, the RV systolic pressure of NFU1G206C rats was significantly (p&lt;0.0001) attenuated by blocking the heme transporter (HCP1) with sulfasalazine (SL, 20mg/ml/kg, i.p.).ConclusionWe show that mitochondrial dysfunction impaired reticulocyte maturation promotes hemolysis resulting in increased CFH. Elevated CFH can trigger pathogenic pathways leading to PH/PAH via EC barrier dysfunction and increased PASMC proliferation. Treatment approaches that improve mitochondrial metabolism (LA) or inhibit free heme entrance to the cell (SL) abrogate the PH phenotype, highlighting the critical role of free heme‐induced signaling in disease pathobiology.

Tendon Healing in a Mouse Model of Loeys-Dietz Syndrome: Controlled Study Using a Patellar Tendon Transection Model.

Loeys-Dietz syndrome (LDS) is a rare autosomal-dominant connective tissue disorder caused by genetic mutations in the transforming growth factor-β (TGFβ) signaling pathway. In addition to vascular malformations, patients with LDS commonly present with bone and tendon abnormalities, including joint laxity. While TGFβ signaling dysregulation has been implicated in many of these clinical manifestations, the degree to which it influences the tendinopathy and tendon healing issues in LDS has not been determined. Wound healing after patellar tendon transection was compared between wild-type (WT) and Tgfbr2-mutant (LDS) mice (7 mice per group). In all mice, the right patellar tendon was transected at midsubstance, while the left was untouched to serve as a control. Mice were euthanized 6 weeks after surgery. Tendon specimens were harvested for histopathologic grading according to a previously validated scoring metric, and gene expression levels of Mmp2, Tgfb2, and other TGFβ-signaling genes were assayed. Between-group comparisons were made using 1-way analysis of variance with post hoc Tukey honestly significant difference testing. Expression levels of assayed genes were similar between LDS and WT tendons at baseline; however, at 6 weeks after patellar tendon transection, LDS tendons showed sustained elevations in Mmp2 and Tgfb2 compared with baseline values; these elevations were not seen in normal tendons undergoing the same treatments. Histologically, untreated LDS tendons had significantly greater cellularity and cell rounding compared with untreated WT tendons, and both WT and LDS tendons had significantly worse histologic scores after surgery. We present the first mechanistic insight into the effect of LDS on tendons and tendon healing. The morphologic differences between LDS and WT tendons at baseline may help explain the increased risk of tendon/ligament dysfunction in patients with LDS, and the differential healing response to injury in LDS may account for the delayed healing and weaker repair tissue. Level V.

Elevated Expression of TLR2 in Aging Hearts Exacerbates Cardiac Inflammatory Response and Adverse Remodeling Following Ischemia and Reperfusion Injury.

This study tested the hypothesis that Toll-like receptor 2 (TLR2) augments the inflammatory responses and adverse remodeling in aging hearts to exacerbate myocardial injury and cardiac dysfunction.MethodsOld (20-22 months old) and adult (4-6 months old) mice of C57BL/6 wild-type and TLR2 knockout (KO) were subjected to coronary artery ligation (30 minutes) and reperfusion (3 or 14 days). Left ventricle function was assessed using a pressure-volume microcatheter. Cardiac infarct size was determined by histology. Levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase 9 (MMP 9), and collagen I in non-ischemic myocardium were assessed by immunoblotting. Monocyte chemoattractant protein-1 (MCP-1), keratinocyte chemoattractant (KC), and interleukin-6 (IL-6) levels in ischemic and non-ischemic myocardium were measured by enzyme-linked immunosorbent assay (ELISA). TLR2 expression in the myocardium of untreated wild type mice was also measured by immunoblotting.ResultsHigher levels of MCP-1, KC, IL-6 were induced in both ischemic and non-ischemic myocardium of old wild type mice at day 3 and 14 following ischemia/reperfusion (I/R) than those of adult wild type mice. The hyper-inflammatory responses to I/R in aging hearts were associated with elevated levels of myocardial TLR2. TLR2 KO markedly down-regulated the expression of MCP-1, KC, IL-6, ICAM-1 and VCAM-1 in aging hearts at day 3 and 14 following I/R. The down-regulated inflammatory activity in aging TLR2 KO hearts was associated with attenuated production of MMP 9 and collagen I at day 14 and resulted in reduced infarct size and improved cardiac function.ConclusionElevated expression of myocardial TLR2 contributes to the mechanism by which aging exacerbates the inflammatory responses, adverse remodeling and cardiac dysfunction following myocardial I/R in aging.

ASK1 Regulates Bleomycin-induced Pulmonary Fibrosis.

Pulmonary fibrosis (PF) is an abnormal remodeling of cellular composition and extracellular matrix that results in histological and functional alterations in the lungs. Apoptosis signal-regulating kinase-1 (ASK1) is a member of the mitogen-activated protein (MAP) kinase family that is activated by oxidative stress and promotes inflammation and apoptosis. Here we show that bleomycin-induced PF is reduced in Ask1 knockout mice (Ask1-/-) compared with wild-type (WT) mice, with improved survival and histological and functional parameters restored to basal levels. In WT mice, bleomycin caused activation of ASK1, p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) in lung tissue, as well as changes in redox indicators (thioredoxin and heme-oxygenase-1), collagen content, and epithelial-mesenchymal transition markers (EMTs). These changes were largely restored toward untreated WT control levels in bleomycin-treated Ask1-/- mice. We further investigated whether treatment of WT mice with an ASK1 inhibitor, selonsertib (GS-4997), during the fibrotic phase would attenuate the development of PF. We found that pharmacological inhibition of ASK1 reduced activation of ASK1, p38, and ERK1/2 and promoted the restoration of redox and EMT indicators, as well as improvements in histological parameters. Our results suggest that ASK1 plays a central role in the development of bleomycin-induced PF in mice via p38 and ERK1/2 signaling. Together, these data indicate a possible therapeutic target for PF that involves an ASK1/p38/ERK1/2 axis.

PGC1α protects against cisplatin-induced skeletal muscle dysfunction

Background and Hypothesis: We and others have shown that chemotherapy promotes skeletal muscle wasting and weakness (i.e., cachexia) by disrupting mitochondrial homeostasis and causing oxidative stress. Peroxisome proliferative-activated receptor gamma coactivator 1-alpha (PGC1α) is a pivotal regulator of mitochondrial biogenesis and is involved in reducing oxidative damage in skeletal muscle. Hence, in the present study we investigated whether overexpression of skeletal muscle PGC1α (mPGC1α) was sufficient to preserve skeletal muscle mass and function in young and old mice treated with cisplatin.&#x0D; Experimental Design or Project Methods: Young (2-month; n = 5) and old (18-month; n = 5-8) male wild type (WT) or mPGC1α transgenic mice were treated with cisplatin (2.5mg/kg), while age-matched WT mice received vehicle for 2 weeks. Animals were assessed for muscle force and motor unit number estimation (MUNE). Skeletal muscles were weighed and processed for molecular analyses, including assessment of mitochondrial protein content.&#x0D; Results: Young WT mice exposed to cisplatin showed evidence of cachexia, as indicated by reduced gastrocnemius size (-16%), plantarflexion force (-8%) and MUNE (-56%), whereas mPGC1α mice were only partially protected. Interestingly, despite exacerbated cachexia in aged WT mice treated with chemotherapy, as demonstrated by markedly decreased gastrocnemius size (-22%), plantarflexion force (-18%) and MUNE (-80%) compared to untreated WT, muscle mass, strength and innervation were fully preserved in age-matched mPGC1α mice. Follow-up molecular analyses revealed that WT animals exposed to chemotherapy present loss of muscle mitochondrial proteins PGC1α, OPA1 and CytochromeC, whereas their levels in mPGC1α mice were robustly increased.&#x0D; Conclusion and Potential Impact: Altogether, our data suggest that PGC1α plays a pivotal role in preserving skeletal muscle mass and function, usually impaired by anticancer treatments. These findings enforce developing mitochondria-targeting therapeutics to combat the negative consequences that chemotherapy has on skeletal muscle.