Increased Superoxide Production Research Articles

The protective effect of imatinib against pancreatic β-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress

Glucocorticoids (GCs) are known to stimulate pancreatic beta (β)-cell apoptosis via several mechanisms, including oxidative stress. Our previous study suggested an increase in dexamethasone-induced pancreatic β-cell apoptosis via a reduction of glutathione S-transferase P1 (GSTP1), which is an antioxidant enzyme. Imatinib, which is a tyrosine kinase inhibitor, also exerts antioxidant effect. This study aims to test our hypothesis that imatinib would prevent pancreatic β-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress. Our results revealed that dexamethasone significantly increased apoptosis in INS-1 cells when compared to the control, and that imatinib significantly decreased INS-1 cell apoptosis induced by dexamethasone. Moreover, dexamethasone significantly increased superoxide production in INS-1 cells when compared to the control; however, imatinib, when combined with dexamethasone, significantly reduced superoxide production in INS-1 cells. Dexamethasone significantly decreased GSTP1, p-ERK1/2, and BCL2 protein expression, but significantly increased p-JNK, p-p38, and BAX protein expression in INS-1 cells—all compared to control. Importantly, imatinib significantly ameliorated the effect of dexamethasone on the expression of GSTP1, p-ERK1/2, p-JNK, p-p38 MAPK, BAX, and BCL2. Furthermore—6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), which is a GSTP1 inhibitor, neutralized the protective effect of imatinib against pancreatic β-cell apoptosis induced by dexamethasone. In conclusion, imatinib decreases pancreatic β-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress.

Chemerin in caudal division of nucleus tractus solitarius increases sympathetic activity and blood pressure.

Chemerin is an adipokine that contributes to metabolism regulation. Nucleus tractus solitarius (NTS) is the first relay station in the brain for accepting various visceral afferent activities for regulating cardiovascular activity. However, the roles of chemerin in the NTS in regulating sympathetic activity and blood pressure are almost unknown. This study aimed to determine the role and potential mechanism of chemerin in the NTS in modulating sympathetic outflow and blood pressure. Bilateral NTS microinjections were performed in anaesthetized adult male Sprague-Dawley rats. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were continuously recorded. Chemerin and its receptor chemokine-like receptor 1 (CMKLR1) were highly expressed in caudal NTS (cNTS). Microinjection of chemerin-9 to the cNTS increased RSNA, MAP and HR, which were prevented by CMKLR1 antagonist α-NETA, superoxide scavenger tempol or N-acetyl cysteine, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenyleneiodonium or apocynin. Chemerin-9 increased superoxide production and NADPH oxidase activity in the cNTS. The increased superoxide production induced by chemerin-9 was inhibited by α-NETA. The effects of cNTS microinjection of chemerin-9 on the RSNA, MAP and HR were attenuated by the pretreatment with paraventricular nucleus (PVN) microinjection of NMDA receptor antagonist MK-801 rather than AMPA/kainate receptor antagonist CNQX. These results indicate that chemerin-9 in the NTS increases sympathetic outflow, blood pressure and HR via CMKLR1-mediated NADPH oxidase activation and subsequent superoxide production in anaesthetized normotensive rats. Glutamatergic inputs in the PVN are needed for the chemerin-9-induced responses.

Unveiling the molecular basis of paracetamol-induced hepatotoxicity: Interaction of N-acetyl-p-benzoquinone imine with mitochondrial succinate dehydrogenase

Background and aimN-acetyl-p-benzoquinoneimine (NAPQI), a toxic byproduct of paracetamol (Acetaminophen, APAP), can accumulate and cause liver damage by depleting glutathione and forming protein adducts in the mitochondria. These adducts disrupt the respiratory chain, increasing superoxide production and reducing ATP. The goal of this study was to provide computational proof that succinate dehydrogenase (SDH), a subunit of complex II in the mitochondrial respiratory chain, is a favorable binding partner for NAPQI in this regard. MethodMolecular docking, molecular dynamics simulation, protein-protein interaction networks (PPI), and KEGG metabolic pathway analysis were employed to identify binding characteristics, interaction partners, and their associations with metabolic pathways. A lipid membrane was added to the experimental apparatus to mimic the natural cellular environment of SDH. This modification made it possible to develop a context for investigating the role and interactions of SDH within a cellular ecosystem that was more realistic and biologically relevant. ResultThe molecular binding affinity score for APAP and NAPQI with SDH was predicted −6.5 and −6.7 kcal/mol, respectively. Furthermore, RMSD, RMSF, and Rog from the molecular dynamics simulations study revealed that NAPQI has slightly higher stability and compactness compared to APAP at 100 ns timeframe with mitochondrial SDH. ConclusionThis study serves to predict the mechanistic process of paracetamol toxicity by using different computational approaches. In addition, this study will provide information about the drug target against APAP hepatotoxicity.

Neutrophil response to Porphyromonas gingivalis is modulated by low-level laser application.

Neutrophil response is critical in inflammatory regulation and immune response to bacterial infections. During periodontal disease, pathogenic bacteria lead to exaggerated neutrophil responses. We hypothesized that low-level laser application (LLLT), therapeutic strategy for dampening inflammatory processes, will regulate neutrophil activity in response to periodontopathogens. The impact of LLLT on neutrophil responses was measured by light delivered at wavelength of 850 nm. The direct effect of LLLT on P. gingivalis A7436 was determined by flow cytometry using LIVE/DEADTM Cell Vitality kit. The phagocytosis of P. gingivalis A7436 by human neutrophils was measured using flow cytometry. Superoxide generation was measured by cytochrome-C-reduction in the presence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 mM). Cytokine release by neutrophils was measured by multiplex immunoassay. The phagocytosis of P. gingivalis by primary human neutrophils was significantly reduced in response to LLLT (p < 0.05). While LLLT led to increased superoxide production in neutrophils that were not challenged by P. gingivalis, it dampened the increased superoxide and IL-6 release by the neutrophils in response to P. gingivalis. LLLT did not directly affect the viability of P. gingivalis. These results suggested that LLLT can provide therapeutic strategy in periodontal disease, regulating the neutrophil response to P. gingivalis.

GLP-1 in the Hypothalamic Paraventricular Nucleus Promotes Sympathetic Activation and Hypertension.

Glucagon-like peptide-1 (GLP-1) and its analogs are widely used for diabetes treatment. The paraventricular nucleus (PVN) is crucial for regulating cardiovascular activity. This study aims to determine the roles of GLP-1 and its receptors (GLP-1R) in the PVN in regulating sympathetic outflow and blood pressure. Experiments were carried out in male normotensive rats and spontaneously hypertensive rats (SHR). Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded. GLP-1 and GLP-1R expressions were present in the PVN. PVN microinjection of GLP-1R agonist recombinant human GLP-1 (rhGLP-1) or EX-4 increased RSNA and MAP, which were prevented by GLP-1R antagonist exendin 9-39 (EX9-39) or GLP-1R antagonist 1, superoxide scavenger tempol, antioxidant N-acetylcysteine, NADPH oxidase (NOX) inhibitor apocynin, adenylyl cyclase (AC) inhibitor SQ22536 or protein kinase A (PKA) inhibitor H89. PVN microinjection of rhGLP-1 increased superoxide production, NADPH oxidase activity, cAMP level, AC, and PKA activity, which were prevented by SQ22536 or H89. GLP-1 and GLP-1R were upregulated in the PVN of SHR. PVN microinjection of GLP-1 agonist increased RSNA and MAP in both WKY and SHR, but GLP-1 antagonists caused greater effects in reducing RSNA and MAP in SHR than in WKY. The increased superoxide production and NADPH oxidase activity in the PVN of SHR were augmented by GLP-1R agonists but attenuated by GLP-1R antagonists. These results indicate that activation of GLP-1R in the PVN increased sympathetic outflow and blood pressure via cAMP-PKA-mediated NADPH oxidase activation and subsequent superoxide production. GLP-1 and GLP-1R upregulation in the PVN partially contributes to sympathetic overactivity and hypertension.

Abstract 4710: The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism

Abstract Background: Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are devastating blood cancers associated with 5-year survival rates of ~50% each in adults. We recently developed a new class of polymeric drugs called PAMDs based on the CXCR4 inhibitor AMD3100 (plerixafor). PAMDs have the potential to be more effective and less toxic therapeutics than the standard of care high-dose chemotherapy. We tested one variant (PAMD-Ch17), and found that unlike AMD3100, PAMD-Ch17 has several novel anti-leukemic effects. To understand the potential mechanism(s) for these new activities, we performed the following study. Methods: To test the role of CXCR4 in PAMD-Ch17’s activities, we generated CXCR4 knockout cells via Crispr/Cas9. We used PrestoBlue, 7-AAD, and Annexin V staining to assay viability and apoptosis. To identify pathways deregulated by PAMD-Ch17, we performed whole transcriptomic sequencing (RNA-Seq). To investigate if PAMD-Ch17 mediates its activities by inducing oxidative stress, we performed SeaHorse assays, and dihydroethidium (DHE), Mitosox, and Bodipy C11 staining. To investigate whether PAMD induces programmed cell death pathways, we used ferrostatin and deferoxamine to inhibit ferroptosis, necrostatin-1 against necroptosis, and Z-VAD-FMK against apoptosis. Results: We found that PAMD-Ch17 induces cell death in a dose dependent manner in human AML and ALL cell lines and, mouse primary leukemia cells, but not healthy mouse bone marrow cells. Surprisingly, PAMD-Ch17 induces equivalent levels of cell death in both wild type and CXCR4 knockout Jurkat cells, indicating that the only known target of the polymer is not required for its anti-leukemic effects. By RNA-Seq, we found altered expression of genes related to mitochondrial function, suggesting that PAMD-Ch17 could be targeting the mitochondria. To test this, we performed SeaHorse assays and found that PAMD-Ch17 induces a significant decrease in mitochondrial respiration. Consistent with this, we found that PAMD-Ch17 induces a significant increase in superoxide production, as well as lipid peroxidation. Using inhibitors of programmed cell death pathways, we found that preventing necroptosis, apoptosis, or ferroptosis caused only subtle effects on PAMD-Ch17 induced cell death, implying that the polymer’s anti-leukemic activity is likely not entirely dependent on these pathways. Conclusions: These results indicate that PAMD-Ch17 induces cell death in leukemia cells but not healthy bone marrow via a CXCR4 independent mechanism. We also found that PAMD-Ch17 induces reactive oxygen species, potentially explaining its selectivity for leukemia cells compared to healthy blood cells. Continued investigation of PAMD-Ch17 will not only further development of this new class of drugs, but also provide much needed insight into leukemia biology. Citation Format: Calvin Lam, Jogdeo Chinmay, Ekta Kapoor, Siyuan Tang, Svetlana Romanova, David Oupicky, Katherine Hyde. The polymeric AMD3100 based drug PAMD-Ch17 induces its anti-leukemic effects in a CXCR4 independent mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4710.

Decreased immune response in undernourished rats after air pollution exposure

Children are highly vulnerable subpopulation to malnutrition and air pollution. We investigate, in a rat nutritional growth retardation (NGR) model, the impact of Residual Oil Fly Ash (ROFA) on the lung immune response using in vitro and ex vivo methods. In vitro: Alveolar macrophages (AM) were isolated from Control (C) and NGR animals, cultured and treated with ROFA (1–100 µg/ml) for 24 h. Ex vivo: C and NGR rats were intranasally instilled with ROFA (1 mg/kg BW) or PBS. 24 h post-exposure AM were isolated and cultured. ROFA-treatment increased superoxide anion production and TNFα secretion in C-AM in vitro, though for NGR-AM this response was lower. A similar pattern was observed for TNFα and IL-6 secretion in ex vivo experiments. Regarding the antioxidant response, although NGR-AM showed increased Nrf2, after ROFA instillation an attenuated activation was observed. To conclude, chronic undernutrition altered AM response to ROFA affecting immune responsiveness to air pollutants.

Elaeagnus umbellata Fruit Extract Protects Skin from Ultraviolet-Mediated Photoaging in Hairless Mice.

Photoaging refers to the accumulation of skin damage which includes wrinkle formation, loss of elasticity, and epidermal thickening due to repeated ultraviolet (UV) irradiation. The present study investigated the protective effects of Elaeagnus umbellata fruit extract (Elaea) on UV-mediated photoaged skin of SKH1 hairless mice and compared the effects of Elaea with ascorbic acid. Although there was no difference in body weight between groups during experimental period, oral administration of 50-200 mg/kg Elaea once daily for 15 weeks significantly prevented an increase in skin weight, epithelial thickening of epidermis, and apoptosis caused by UV irradiation. Skin replica and histopathological analyses revealed that Elaea dose-dependently decreased wrinkle and microfold formation. In addition, Elaea administration restored UV-mediated reduction in type I collagen and hyaluronan through the inhibition of matrix metalloproteinases and p38 mitogen-activated protein kinase expression. Moreover, Elaea suppressed UV-dependent increases in superoxide anion production, fatty acid oxidation, and protein nitration by up-regulating antioxidant system. Furthermore, Elaea alleviated infiltration of inflammatory cells in UV-irradiated skin. The preventive effects of 100 mg/kg Elaea administration against UV-induced photoaging were similar to those by 100 mg/kg ascorbic acid. Collectively, the present study suggests that the E. umbellata fruit is a promising edible candidate to prevent skin photoaging.

Prostaglandin E2 affects mitochondrial function in adult mouse cardiomyocytes and hearts

Prostaglandin E2 (PGE2) signals differently through 4 receptor subtypes (EP1-EP4) to elicit diverse physiologic/pathologic effects. We previously reported that PGE2 via its EP3 receptor reduces cardiac contractility and male mice with cardiomyocyte-specific deletion of the EP4 receptor (EP4 KO) develop dilated cardiomyopathy. The aim of this study was to identify pathways responsible for this phenotype. We performed ingenuity pathway analysis (IPA) and found that genes differentiating WT mice and EP4 KO mice were significantly overrepresented in mitochondrial (adj. p value = 6.28 × 10−26) and oxidative phosphorylation (adj. p value = 1.58 × 10−27) pathways. Electron microscopy from the EP4 KO hearts show substantial mitochondrial disarray and disordered cristae. Not surprisingly, isolated adult mouse cardiomyocytes (AVM) from these mice have reduced ATP levels compared to their WT littermates and reduced expression of key genes involved in the electron transport chain (ETC) in older mice. Moreover, treatment of AVM from C57Bl/6 mice with PGE2 or the EP3 agonist sulprostone resulted in changes of various genes involved in the ETC, measured by the Mitochondrial Energy Metabolism RT2-profiler assay. Lastly, the EP4 KO mice have reduced expression of superoxide dismuatse-2 (SOD2), whereas treatment of AVM with PGE2 or sulprostone increase superoxide production, suggesting increased oxidative stress levels in these EP4 KO mice. Altogether the current study supports the premise that PGE2 acting via its EP4 receptor is protective, while signaling through its other receptors, likely EP3, is deleterious.

Demonstrating drug treatment efficacies by monitoring superoxide dynamics in human lung cancer cells with time-lapse fluorescence microscopy.

Metformin hydrochloride, an antihyperglycemic agent, and sulindac, a nonsteroidal anti-inflammatory drug, are FDA-approved drugs known to exert anticancer effects. Previous studies demonstrated sulindac and metformin's anticancer properties through mitochondrial dysfunction and inhibition of mitochondrial electron transport chain complex I and key signaling pathways. In this study, various drugs were administered to A549 lung cancer cells, and results revealed that a combination of sulindac and metformin enhanced cell death compared to the administration of the drugs separately. To measure superoxide production over time, we employed a time-lapse fluorescence imaging technique using mitochondrial-targeted hydroethidine. Fluorescence microscopy data showed the most significant increases in superoxide production in the combination treatment of metformin and sulindac. Results showed significant differences between the combined drug treatment and control groups and between the positive control and control groups. This approach can be utilized to quantify the anticancer efficacy of drugs, creating possibilities for additional therapeutic options.

Co-exposure to cadmium and microplastics promotes liver fibrosis through the hemichannels -ATP-P2X7 pathway

Microplastics (MPs) and cadmium (Cd) are important environmental pollutants, that damage the liver. However, the effect and mechanism of combined Cd and MPs exposure on liver fibrosis are still largely unknown. In this study investigated, Cd + MPs exposure increased superoxide anion production and promoted extracellular ATP release compared with exposure to Cd or MPs individually. Cd + MPs increased inflammatory cell infiltration, activated the P2X7-NLRP3 signaling pathway, and promoted inflammatory factor release. Cd + MPs aggravated Cd- or MPs-induced liver fibrosis and induced liver inflammation. In AML12/HSC-T6 cell in vitro poisoning model, exposure of AML12 cells to Cd + MPs increased the opening of connexin hemichannels and promoted extracellular ATP release. Treatment of HSC-T6 cells with the supernatant of AML12 cells exposed to Cd + MPs significantly promoted HSC-T6 cell activation. Treatment of HSC-T6 cells with different concentrations of ATP produced similar results. TAT-Gap19TFA, an inhibitor of connexin hemichannels, significantly inhibited the ATP release and activation of Cd + MPs-treated HSC-T6 cells. Finally, the expression of the ATP receptor P2X7 was silenced in HSC-T6 cells, which significantly inhibited their activation. In conclusion, exposure to Cd + MPs promoted liver fibrosis through the ATP-P2X7 pathway and synergistically affected liver inflammation and fibrosis.

Telomerase reverse transcriptase restores pancreatic microcirculation profiles and attenuates endothelial dysfunction by inhibiting mitochondrial superoxide production: A potential target for acute pancreatitis therapy

BackgroundAcute pancreatitis (AP) is a potentially lethal disease related to prominent microcirculation dysfunction. Pancreatic microvascular endothelial dysfunction enhances oxidative stress with tissue damage. Increased superoxide production disrupts endothelial junction integrity and increases endothelial permeability. Endothelial mitochondrial ROS (mtROS) represent a major intracellular source of superoxide anions. The non-canonical function of telomerase reverse transcriptase (TERT) involves the maintenance of cellular redox homeostasis in somatic tissues. MethodsWe investigated whether TERT restores microcirculation dysfunction and attenuates the endothelium injury by inhibiting superoxide production during AP progression. We established TERT transgenic and TERT knock-down mice and used cerulein (CER) and lipopolysaccharide (LPS) injections to induce AP models. In addition, we exposed HUVECs to LPS following TERT overexpression or silencing to explore the role of TERT in endothelial dysfunction. We also performed flow cytometry and confocal microscopy assays by using HUVECs. And a mtROS inhibitor, MitoTempo, was used to scavenge mitochondria superoxide and alkyl. ResultsTERT transgenic mice were found to have restored pancreatic microcirculation profiles and microvascular endothelial morphology compared with wild-type mice under cerulein injection. In contrast, TERT silencing displayed the opposite effect in response to cerulein. Subsequently, we showed that TERT overexpression attenuates mtROS production and mitochondrial dysfunction during LPS-stimulated endothelial dysfunction. Furthermore, we found that TERT overexpression maintains the balance between mitochondrial contents and ATP level during endothelial dysfunction. In addition, the protective trend of MitoTempo is impeded after TERT silencing. ConclusionTERT restores pancreatic microcirculation dysfunction and attenuates microvascular endothelium lesions by inhibiting the increase of superoxide production and mitochondrial dysfunction.

Glucose fluctuation promotes mitochondrial dysfunctions in the cardiomyocyte cell line HL-1.

Glycemic variability has been suggested as a risk factor for diabetes complications but the precise deleterious mechanisms remain poorly understood. Since mitochondria are the main source of energy in heart and cardiovascular diseases remain the first cause of death in patients with diabetes, the aim of the study was to evaluate the impact of glucose swings on mitochondrial functions in the cardiomyocyte cell line HL-1. HL-1 cells were exposed to low (LG, 2.8 mmol/l), normal (NG, 5.5 mmol/l), high (HG, 25 mmol/l) or intermittent high glucose (IHG, swing between low and high) every 2h during 12h (short-time treatment) or every 12h during 72h (long-time treatment). Anaerobic catabolism of glucose was evaluated by measuring glucose consumption and lactate production, oxidative phosphorylation was evaluated by polarography and ATP measurement, mitochondrial superoxide anions and the mitochondrial membrane potential (MMP) were analysed using fluorescent probes, and the protein oxidation was measured by oxyblot. IHG and HG increased glucose consumption and lactate production compared to LG and NG but without any difference between short- and long-time treatments. After 72h and unlike to LG, NG and HG, we didn't observe any increase of the mitochondrial respiration in the presence of succinate upon IHG treatment. IHG, and to a lesser extent HG, promoted a time-dependent decrease of the mitochondrial membrane potential compared to LG and NG treatments. HG and IHG also increased superoxide anion production compared to LG and NG both at 12 and 72h but with a higher increase for IHG at 72h. At last, both HG and IHG stimulated protein oxidation at 72h compared to LG and NG treatments. Our results demonstrated that exposure of HL-1 cells to glucose swings promoted time-dependent mitochondrial dysfunctions suggesting a deleterious effect of such condition in patients with diabetes that could contribute to diabetic cardiomyopathy.

Extracellular pyruvate kinase M2 induces cell migration through p-Tyr42 RhoA-mediated superoxide generation and epithelial-mesenchymal transition

In the tumor microenvironment (TME), communication between cancer cells and tumor-associated macrophages (TAMs) through secreted extracellular proteins promotes cancer progression. Here, we observed that co-culturing cancer cells (4T1) and macrophage cells (Raw264.7) significantly enhanced superoxide production in both cell types. Using MALDI-TOF, we identified PKM2 as a highly secreted protein by Raw264.7 cells and bone marrow-derived monocytes. The extracellular recombinant PKM2 protein not only enhanced cancer cell migration and invasion but also increased superoxide production. Additionally, PKM2 was found to associate with the cell surface, and its binding to integrin α5/β1 receptor was inhibited by antibodies specifically targeting it.Furthermore, we investigated downstream signaling pathways involved in PKM2-induced superoxide production. We found that knock-down of RhoA and p47phox using siRNAs effectively abolished superoxide generation in response to extracellular PKM2. Notably, extracellular PKM2 triggered the phosphorylation of p47phox at Ser345 residue and RhoA at Tyr42 residue (p-Tyr42 RhoA). Moreover, extracellular PKM2 exerted regulatory control over the expression of key epithelial-mesenchymal transition (EMT) markers, including ZEB1, Snail1, vimentin, and E-cadherin. Interestingly, p-Tyr42 RhoA translocated to the nucleus, where it bound to the ZEB1 promoter region. In light of these findings, we propose that extracellular PKM2 within the TME plays a critical role in tumorigenesis by promoting cancer cell migration and invasion through RhoA/p47phox signaling pathway.

Association of Apolipoprotein E4-related Microvascular Disease in the Alzheimer’s Disease Hippocampal CA1 Stratum Radiatum

Current data suggest a hypothesis of vascular pathogenesis for the development and progression of Alzheimer’s disease (AD). To investigate this, we studied the association of apolipoprotein E4 (APOE4) gene on microvessels in human autopsy-confirmed AD with and without APOE4, compared with age/sex-matched control (AC) hippocampal CA1 stratum radiatum. AD arterioles (without APOE4 gene) had mild oxidative stress and loss of vascular endothelial growth factor (VEGF) and endothelial cell density, reflecting aging progression. In AD + APOE4, an increase in strong oxidative DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OHdG), VEGF, and endothelial cell density were associated with increased diameter of arterioles and perivascular space dilation. In cultured human brain microvascular cells (HBMECs), treatment of ApoE4 protein plus amyloid-β (Aβ) oligomers increased superoxide production and the apoptotic marker cleaved caspase 3, sustained hypoxia inducible factor-1α (HIF-1α) stability that was associated with an increase in MnSOD, VEGF, and cell density. This cell over-proliferation was inhibited with the antioxidants N-acetyl cysteine and MnTMPyP, the HIF-1α inhibitor echinomycin, the VEGFR-2 receptor blocker SU1498, the protein kinase C (PKC) ε knock-down (KD) and the extracellular signal-regulated kinase 1/2 (ERK) inhibitor FR180204. The PKCε KD and echinomycin decreased VEGF and/or ERK. In conclusion, AD capillaries and arterioles in hippocampal CA1 stratum radiatum of non-APOE4 carriers are related with aging, while those in APOE4 carriers with AD are related with pathogenesis of cerebrovascular disease.

Absence of TRIC-B from type XIV Osteogenesis Imperfecta osteoblasts alters cell adhesion and mitochondrial function – A multi-omics study

Osteogenesis Imperfecta (OI) is a heritable collagen-related bone dysplasia characterized by bone fractures, growth deficiency and skeletal deformity. Type XIV OI is a recessive OI form caused by null mutations in TMEM38B, which encodes the ER membrane intracellular cation channel TRIC-B. Previously, we showed that absence of TMEM38B alters calcium flux in the ER of OI patient osteoblasts and fibroblasts, which further disrupts collagen synthesis and secretion. How the absence of TMEM38B affects osteoblast function is still poorly understood. Here we further investigated the role of TMEM38B in human osteoblast differentiation and mineralization. TMEM38B-null osteoblasts showed altered expression of osteoblast marker genes and decreased mineralization. RNA-Seq analysis revealed that cell-cell adhesion was one of the most downregulated pathways in TMEM38B-null osteoblasts, with further validation by real-time PCR and Western blot. Gap and tight junction proteins were also decreased by TRIC-B absence, both in patient osteoblasts and in calvarial osteoblasts of Tmem38b-null mice. Disrupted cell adhesion decreased mutant cell proliferation and cell cycle progression. An important novel finding was that TMEM38B-null osteoblasts had elongated mitochondria with altered fusion and fission markers, MFN2 and DRP1. In addition, TMEM38B-null osteoblasts exhibited a significant increase in superoxide production in mitochondria, further supporting mitochondrial dysfunction. Together these results emphasize the novel role of TMEM38B/TRIC-B in osteoblast differentiation, affecting cell-cell adhesion processes, gap and tight junction, proliferation, cell cycle, and mitochondrial function.

Heme catabolism mediated by heme oxygenase in uninfected interstitial cells enables efficient symbiotic nitrogen fixation in Lotus japonicus nodules.

Legume nodules produce large quantities of heme required for the synthesis of leghemoglobin (Lb) and other hemoproteins. Despite the crucial function of Lb in nitrogen fixation and the toxicity of free heme, the mechanisms of heme homeostasis remain elusive. Biochemical, cellular, and genetic approaches were used to study the role of heme oxygenases (HOs) in heme degradation in the model legume Lotus japonicus. Heme and biliverdin were quantified and localized, HOs were characterized, and knockout LORE1 and CRISPR/Cas9 mutants for LjHO1 were generated and phenotyped. We show that LjHO1, but not the LjHO2 isoform, is responsible for heme catabolism in nodules and identify biliverdin as the invivo product of the enzyme in senescing green nodules. Spatiotemporal expression analysis revealed that LjHO1 expression and biliverdin production are restricted to the plastids of uninfected interstitial cells. The nodules of ho1 mutants showed decreased nitrogen fixation, and the development of brown, rather than green, nodules during senescence. Increased superoxide production was observed in ho1 nodules, underscoring the importance of LjHO1 in antioxidant defense. We conclude that LjHO1 plays an essential role in degradation of Lb heme, uncovering a novel function of nodule plastids and uninfected interstitial cells in nitrogen fixation.

Constitutive and induced forms of membrane-bound proteinase 3 interact with antineutrophil cytoplasmic antibodies and promote immune activation of neutrophils

Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFα-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigen-binding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis.

Neurovascular Coupling in Hypertension Is Impaired by IL-17A through Oxidative Stress.

Hypertension, a multifactorial chronic inflammatory condition, is an important risk factor for neurovascular and neurodegenerative diseases, including stroke and Alzheimer's disease. These diseases have been associated with higher concentrations of circulating interleukin (IL)-17A. However, the possible role that IL-17A plays in linking hypertension with neurodegenerative diseases remains to be established. Cerebral blood flow regulation may be the crossroads of these conditions because regulating mechanisms may be altered in hypertension, including neurovascular coupling (NVC), known to participate in the pathogenesis of stroke and Alzheimer's disease. In the present study, the role of IL-17A on NVC impairment induced by angiotensin (Ang) II in the context of hypertension was examined. Neutralization of IL-17A or specific inhibition of its receptor prevents the NVC impairment (p < 0.05) and cerebral superoxide anion production (p < 0.05) induced by Ang II. Chronic administration of IL-17A impairs NVC (p < 0.05) and increases superoxide anion production. Both effects were prevented with Tempol and NADPH oxidase 2 gene deletion. These findings suggest that IL-17A, through superoxide anion production, is an important mediator of cerebrovascular dysregulation induced by Ang II. This pathway is thus a putative therapeutic target to restore cerebrovascular regulation in hypertension.

ACTA1 H40Y mutant iPSC-derived skeletal myocytes display mitochondrial defects in an in vitro model of nemaline myopathy

Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study.