Examining the role of nickel and NiTi nanoparticles promoting inflammation and angiogenesis.

Nickel titanium (NiTi, or Nitinol) alloy is used in several biomedical applications, including cardiac, peripheral vascular, and fallopian tube stents. There are significant biocompatibility issues of metallic implants to nickel ions and nano-/micro-sized alloy particles. Our laboratories have recently shown that microscale CoCr wear particles from metal-on-metal hips crosslink with the innate immune signaling Toll-like receptor 4 (TLR4), prompting downstream signaling that results in interleukin (IL)-1ß and IL-8 gene expression. In vivo, NiTi alloy can also generate wear particles on the nanoscale (NP) that have thus far not been studied for their potential to induce inflammation and angiogenesis that can, in turn, contribute to implant (e.g. stent) failure. Earlier studies by others demonstrated that nickel could induce contact hypersensitivity by crosslinking the human, but not the mouse, TLR4. In the present work, it is demonstrated that NiCl2 ions and NiTi nanoparticles induce pro-inflammatory and pro-angiogenic cytokine/chemokine expression in human endothelial and monocyte cell lines in vitro. These observations prompt concerns about potential mechanisms for stent failure. The data here showed a direct correlation between intracellular uptake of Ni2+ and generation of reactive oxygen species. To determine a role for nickel and NiTi nanoparticles in inducing angiogenesis in vivo, 1-cm silicone angioreactors were implanted subcutaneously into athymic (T-cell-deficient) nude mice. The angioreactors contained Matrigel (a gelatinous protein mixture that resembles extracellular matrix) in addition to one of the following: PBS (negative control), VEGF/FGF-2 (positive control), NiCl2, or NiTi NP. The implantation of angioreactors represents a potential tool for quantification of angiogenic potentials of medical device-derived particles and ions in vivo. By this approach, NiTi NP were found to be markedly angiogenic, while Ni2+ was less-so. The angioreactors may provide a powerful tool to examine if debris shed from medical devices may promote untoward biological effects.

Neuronal Bmi-1 is critical for melatonin induced ubiquitination and proteasomal degradation of α-synuclein in experimental Parkinson's disease models.

Epigenetic polycomb repressor complex-1 subunit BMI-1 plays a pivotal role in the process of gene repression to maintain the self-renewal and differentiation state of neurogenic tissues. Accumulating reports links lower expression of BMI-1 fails to regulate the repression of anti-oxidant response genes disrupt mitochondrial homeostasis underlying neurodegeneration. Interestingly, this negative relation between BMI-1 function and neurodegeneration is distinct but has not been generalized as a potential biomarker particularly in Parkinson's disease (PD). Hyperphosphorylated BMI-1 undergoes canonical polycomb E3 ligase function loss, thereby leads to reduce monoubiquitylation of histone 2A at lysine 119 (H2AK119ub) corroborates cellular accumulation of α-synuclein protein phosphorylated at serine 129 (pα-SYN (S129). In general, neuroprotectant suppressing pα-SYN (S129) level turns ineffective upon depletion of neuronal BMI-1. However, it has been observed that our neuroprotectant exposure suppresses the cellular pα-SYN (S129) and restore the the BMI-1 expression level in neuronal tissues. The pharmacological inhibition and activation of proteasomal machinery promote the cellular accumulation and degradation of neuronal pα-SYN (S129), respectively. Furthermore, our investigation reveals that accumulated pα-SYN (S129) are priorly complexed with BMI-1 undergoes ubiquitin-dependent proteasomal degradation and established as key pathway for therpeutic effect in PD. These findings linked the unestablished non-canonical role of BMI-1 in the clearance of pathological α-SYN and suspected to be a novel therapeutic target in PD.

A non-viral nano-delivery system targeting epigenetic methyltransferase EZH2 for precise acute myeloid leukemia therapy.

Acute myeloid leukemia (AML), which is common in the elderly population, accounts for poor long-term survival with a high possibility of relapse. The associated lack of currently developed therapeutics is directing the search for new therapeutic targets relating to AML. EZH2 (Enhancer of Zeste Homolog 2) is a histone methyltransferase member of the polycomb-group (PcG) family, and its significant overexpression in AML means it has emerged as a potential epigenetic target. Here, we propose the human serum albumin (HSA) nanoparticle based delivery of small interfering RNA (siRNA), which can target EZH2-expressing genes in AML. EZH2 specific siRNA loaded in a polyethyleneimine (PEI) conjugated HSA nanocarrier can overcome the systemic instability of siRNA and precisely target the AML cell population for increased EZH2 gene silencing. A stable nanosized complex (HSANPs-PEI@EZH2siRNA), achieved via the electrostatic interaction of PEI and EZH2 siRNA, shows increased systemic stability and hemocompatibility, and enhanced EZH2 gene silencing activity in vitro, compared to conventional transfection reagents. HSANPs-PEI@EZH2siRNA-treated AML cells showed downregulated EZH2, which is associated with a reduced level of Bmi-1 protein, and H3K27me3 and H2AK119ub modification. The ubiquitin-mediated proteasomal degradation pathway plays a critical role in the downregulation of associated proteins following HSANPs-PEI@EZH2siRNA exposure to AML cells. c-Myb is the AML-responsive transcription factor that directly binds on the EZH2 promoter and was downregulated in HSANPs-PEI@EZH2siRNA-treated AML cells. The systemic exposure to HSANPs-PEI@EZH2siRNA of AML engrafted immunodeficient nude mice displayed efficient EZH2 gene silencing and a reduced AML cell population in peripheral blood and bone marrow. The present study demonstrates a non-viral siRNA delivery system for epigenetic targeting based superior anti-leukemic therapy.

Nanoformulation of EPZ011989 Attenuates EZH2-c-Myb Epigenetic Interaction by Proteasomal Degradation in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic progenitor cells with a poor prognosis of 26% of patients surviving 5 years after diagnosis. Poor bioavailability and solubility are significant factors limiting the efficacy of chemopreventive agents. In AML, the epigenetic regulator polycomb group of protein member EZH2 is highly expressed and is essential for the survival of leukemic cells. An EZH2-specific inhibitor, EPZ011989, encapsulated in human serum albumin nanoparticles (HSANPs) was synthesized for the first time via the desolvation method. The noncovalent interactions between EPZ011989 and HSANPs in nanocomposites facilitating the efficient loading and sustainable release of the drug showed enhanced cellular uptake and nuclear localization of EPZ011989-loaded HSANPs in human AML cell lines. The reduction of cell viability, colony formation inhibition, cell cycle arrest at the G2/M phase, and cell proliferation assay promoting apoptosis through the loss of mitochondrial homeostasis exerting antileukemic activity were evident. The real-time polymerase chain reaction (PCR) and western blot-based studies showed that the present nanoformulation reduces the level of PcG proteins, including EZH2, BMI-1, etc. This downregulation is associated with reduced H3K27me3 and H2AK119ub modifications conferring chromatin compaction. The immunoprecipitation study showed the physical interaction of EZH2 and c-Myb can be linked to the regulation of leukemogenesis. Further investigation revealed the mechanism of EZH2 and c-Myb downregulation via ubiquitination and proteasomal degradation pathway, confirmed by using proteasome inhibitor, suggesting the key role of proteasomal degradation machinery. Moreover, c-Myb interacted with the EZH2 promoter, which is evident by the chromatin immunoprecipitation assay and siRNA silencing. Furthermore, the formulation of EPZ011989 in HSANPs improved its biodistribution in vivo and showed excellent aqueous dispersibility and biocompatibility. In vivo studies further showed that EPZ011989-loaded HSANPs reduce the expression of CD11b+ and CD45+ markers in immunophenotyping from peripheral blood and bone marrow in engrafted nude mice. Targeted depletion of EZH2 alleviated the disease progression in nude mice and prolonged their survival. The findings provide valuable experimental evidence for the targeted epigenetic therapy of AML. The present results demonstrate an epigenetic regulation-based superior antileukemic therapy.

Melatonin/polydopamine nanostructures for collective neuroprotection-based Parkinson's disease therapy.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and localized deposition of cytoplasmic fibrillary inclusions as Lewy bodies in the brain. The aberrant phosphorylation of α-synuclein at serine 129 is the key process on its early onset, which alters the cellular conformation to oligomers and insoluble aggregates, underpinning cellular oxidative stress and mitochondrial dysfunction, leading to devastating PD synucleinopathy. The multiple neuroprotective roles of dopamine and melatonin are often demonstrated separately; however, this approach suffers from low and short bioavailability and is associated with side-effects upon overdosing. Herein, highly pleiotropic melatonin-enriched polydopamine nanostructures were fabricated, which showed efficient brain tissue retention, sustainable and prolonged melatonin release, and prevented neuroblastoma cell death elicited by Parkinson's disease-associated and mitochondrial damaging stimuli. The synergistic neuroprotection re-established the mitochondrial membrane potential, reduced the generation of cellular reactive oxygen species (ROS), inhibited the activation of both the caspase-dependent and independent apoptotic pathways, and exhibited an anti-inflammatory effect. At the molecular level, it suppressed α-synuclein phosphorylation at Ser 129 and reduced the cellular deposition of high molecular weight oligomers. The therapeutic assessment on ex vivo organotypic brain slice culture, and in vivo experimental PD model confirmed the superior brain targeting, collective neuroprotection on dopaminergic neurons with reduced alpha-synuclein phosphorylation and deposition in the hippocampal and substantia nigra region of the brain. Thus, nature-inspired melatonin-enriched polydopamine nanostructures conferring collective neuroprotective effects attributes activation of anti-oxidative, anti-inflammatory, and anti-apoptotic pathways may be superior for application in a nanomedicine-based PD therapy.

Near-Infrared Responsive Dopamine/Melatonin-Derived Nanocomposites Abrogating in Situ Amyloid ß Nucleation, Propagation, and Ameliorate Neuronal Functions.

Alzheimer's disease (AD) is one of the common causes of dementia and mild cognitive impairments, which is progressively expanding among the elderly population worldwide. A short Amyloid-ß (Aß) peptide generated after amyloidogenic processing of amyloid precursor protein exist as intermolecular ß-sheet rich oligomeric, protofibriler, and fibrillar structures and believe to be toxic species which instigate neuronal pathobiology in the brain and deposits as senile plaque. Enormous efforts are being made to develop an effective anti-AD therapy that can target Aß processing, aggregation, and propagation and provide a synergistic neuroprotective effect. However, a nanodrug prepared from natural origin can confer a multimodal synergistic chemo/photothermal inhibition of Aß pathobiology is not yet demonstrated. In the present work, we report a dopamine-melatonin nanocomposite (DM-NC), which possesses a synergistic near-infrared (NIR) responsive photothermal and pharmacological modality. The noncovalent interaction-mediated self-assembly of melatonin and dopamine oxidative intermediates leads to the evolution of DM-NCs that can withstand variable pH and peroxide environment. NIR-activated melatonin release and photothermal effect collectively inhibit Aß nucleation, self-seeding, and propagation and can also disrupt the preformed Aß fibers examined using in vitro Aß aggregation and Aß-misfolding cyclic amplification assays. The DM-NCs display a higher biocompatibility to neuroblastoma cells, suppress the AD-associated generation of intracellular reactive oxygen species, and are devoid of any negative impact on the axonal growth process. In okadaic acid-induced neuroblastoma and ex vivo midbrain slice culture-based AD model, DM-NCs exposure suppresses the intracellular Aß production, aggregation, and accumulation. Therefore, this nature-derived nanocomposite demonstrates a multimodal NIR-responsive synergistic photothermal and pharmacological modality for effective AD therapy.

Recuperative effect of metformin loaded polydopamine nanoformulation promoting EZH2 mediated proteasomal degradation of phospho-α-synuclein in Parkinson's disease model.

Posttranslational modification and agglomeration of α-synuclein (α-Syn), mitochondrial dysfunction, oxidative stress and loss of dopaminergic neurons are hallmark of Parkinson's disease (PD). This paper evaluates neuroprotection efficacy of nature inspired biocompatible polydopamine nanocarrier for metformin delivery (Met encapsulated PDANPs) by crossing blood brain barrier in in vitro, 3D and in vivo experimental PD models. The neuroprotective potential was arbitrated by downregulation of phospho-serine 129 (pSer129) α-Syn, with reduction in oxidative stress, prevention of apoptosis and anti-inflammatory activities. The neuroprotective mechanism proved novel interaction of epigenetic regulator EZH2 mediated ubiquitination and proteasomal degradation of aggregated pSer129 α-Syn. In summary, this study divulges the neuroprotective role of Met loaded PDANPs by reversing the neurochemical deficits by confirming an epigenetic mediated nanotherapeutic approach for the PD prevention.

Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in Pancreatic Ductal Adenocarcinoma (PDA): An integrative analysis of a novel therapeutic target.

We investigated biomarker CEACAM6, a highly abundant cell surface adhesion receptor that modulates the extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDA). The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) RNA-Seq data from PDA patients were analyzed for CEACAM6 expression and evaluated for overall survival, association, enrichment and correlations. A CRISPR/Cas9 Knockout (KO) of CEACAM6 in PDA cell line for quantitative proteomics, mitochondrial bioenergetics and tumor growth in mice were conducted. We found CEACAM6 is over-expressed in primary and metastatic basal and classical PDA subtypes. Highest levels are in classical activated stroma subtype. CEACAM6 over-expression is universally a poor prognostic marker in KRAS mutant and wild type PDA. High CEACAM6 expression is associated with low cytolytic T-cell activity in both basal and classical PDA subtypes and correlates with low levels of T-REG markers. In HPAF-II cells knockout of CEACAM6 alters ECM-cell adhesion, catabolism, immune environment, transmembrane transport and autophagy. CEACAM6 loss increases mitochondrial basal and maximal respiratory capacity. HPAF-II CEACAM6-/- cells are growth suppressed by >65% vs. wild type in mice bearing tumors. CEACAM6, a key regulator affects several hallmarks of PDA including the fibrotic reaction, immune regulation, energy metabolism and is a novel therapeutic target in PDA.

A NIR-responsive indocyanine green-genistein nanoformulation to control the polycomb epigenetic machinery for the efficient combinatorial photo/chemotherapy of glioblastoma.

Combinatorial photodynamics and chemotherapy have drawn enormous attention as therapeutic modalities via precise stimuli-responsive drug delivery for glioblastoma, which can overcome the limitations associated with conventional therapies. Herein, we have prepared an indocyanine green tagged, genistein encapsulated casein nanoformulation (ICG-Gen@CasNPs) that exhibits the near infra-red region responsive controlled release of genistein and enhanced cellular uptake in the human glioblastoma monolayer and a three-dimensional raft culture model via the enhanced retention effect. ICG-Gen@CasNPs, with the integrated photosensitizer indocyanine green within the nanoformulation, triggered oxidative stress, activating the apoptosis cascade, promoting cell cycle arrest and damaging the mitochondrial membrane potential, collectively directing glioblastoma cell death. The suppression of the polycomb group of proteins in the glioblastoma upon ICG-Gen@CasNPs/NIR exposure revealed the involvement of the epigenetic repression complex machinery in the regulation. Furthermore, ICG-Gen@CasNPs/PDT/PTT directed ubiquitination and proteasomal degradation of EZH2 and BMI1 indicates the implication of the polycomb in conferring glioblastoma survival. The increased activation of the apoptotic pathways and the generation of cellular reactive oxygen species upon inhibiting the expression of EZH2 and BMI1 strengthen our observations. It is worth noting that ICG-Gen@CasNPs robustly accumulated in the brain after crossing the blood-brain barrier, which represents the eminent biocompatibility and means that the system is devoid of any nonspecific toxicity in vivo. Moreover, a superior anti-tumor effect was demonstrated on a three-dimensional glioma spheroid model. Thus, this combinatorial chemo/photodynamic therapy revealed that ICG-Gen@CasNPs mediated epigenetic regulation, which is a crucial molecular mechanism of GBM suppression.

1, 3ß-Glucan anchored, paclitaxel loaded chitosan nanocarrier endows enhanced hemocompatibility with efficient anti-glioblastoma stem cells therapy.

Recurrence of glioblastoma is one of the major concerns due to its heterogeneous nature and association of Glioma Initiating stem-like Cells (GICs). Nanoparticles mediated delivery of chemotherapeutic agent targeting both cancer and glioma stem cells could provide a solution to recurrent malignancies of the glioblastoma tumor. The approach described here provides enhanced chemotherapeutic potency utilizing 1,3ß-Glucan as an outer shell to the chitosan nanoparticles (Cs-NPs) loaded with paclitaxel to prevent hemolysis with, the core-shell nano-structure (Cs-PTX-NP) enabling effective chemotherapy against malignant glioblastoma. The prepared nanoparticles (1,3ß-Cs-PTX-NPs) with sustained release of the paclitaxel provide a targeted therapeutic approach that overcome systemic toxicities with the 1,3ß-Glucan shell and improve drug bioavailability. Hemolysis investigation indicated that 1,3ß-Cs-PTX-NP was significantly less hemolytic than paclitaxel enabling intravenous delivery. Also, 1,3ß-Cs-PTX-NPs were considerably more cytotoxic (IC50) against glioma cancer LN18 cells and C6 stem-like cells compared with the PTX. In conclusion, this study found that 1,3ß-Cs-PTX-NP addressed serious limitation with systemic delivery of paclitaxel by preventing hemolysis and providing chemotherapeutic delivery with significant anti-cancer efficacy against recurrent glioblastoma.

Nanomelatonin triggers superior anticancer functionality in a human malignant glioblastoma cell line.

Melatonin (MEL) has promising medicinal value as an anticancer agent in a variety of malignancies, but there are difficulties in achieving a therapeutic dose due to its short half-life, low bioavailability, poor solubility and extensive first-pass metabolism. In this study chitosan/tripolyphosphate (TPP) nanoparticles were prepared by an ionic gelation method to overcome the therapeutic challenges of melatonin and to improve its anticancer efficacy. Characterization of the melatonin-loaded chitosan (MEL-CS) nanoformulation was performed using transmission and scanning electron microscopies, dynamic light scattering, Fourier transform infrared spectroscopy, Raman spectroscopy and x-ray diffraction. In vitro release, cellular uptake and efficacy studies were tested for their enhanced anticancer potential in human U87MG glioblastoma cells. Confocal studies revealed higher cellular uptake of MEL-CS nanoparticles and enhanced anticancer efficacy in human malignant glioblastoma cancer cells than in healthy non-malignant human HEK293T cells in mono- and co-culture models. Our study has shown for the first time that MEL-CS nanocomposites are therapeutically more effective as compared to free MEL at inducing functional anticancer efficacy in the human brain tumour U87MG cell line.

Proteomics data on MAP Kinase Kinase 3 knock out bone marrow derived macrophages exposed to cigarette smoke extract.

This data article reports changes in the phosphoproteome and total proteome of cigarette smoke extract (CSE) exposed WT and MAP Kinase Kinase 3 knock out (MKK3-/-) bone marrow derived macrophages (BMDM). The dataset generated is helpful for understanding the mechanism of CSE induced inflammation and the role of MAP kinase signaling pathway. The cellular proteins were labeled with isobaric tags for relative and absolute quantitation (iTRAQ®) reagents and analyzed by LC-MS/MS. The standard workflow module for iTRAQ® quantification within the Proteome Discoverer was utilized for the data analysis. Ingenuity Pathway Analysis (IPA) software and Reactome was used to identify enriched canonical pathways and molecular networks (Mannam et al., 2016) [1]. All the associated mass spectrometry data has been deposited in the Yale Protein Expression Database (YPED) with the web-link to the data: http://yped.med.yale.edu/repository/ViewSeriesMenu.do;jsessionid=6A5CB07543D8B529FAE8C3FCFE29471D?series_id=5044&series_name=MMK3+Deletion+in+MEFs.

MKK3 influences mitophagy and is involved in cigarette smoke-induced inflammation.

Cigarette smoking is the primary risk factor for COPD which is characterized by excessive inflammation and airflow obstruction of the lung. While inflammation is causally related to initiation and progression of COPD, the mitochondrial mechanisms that underlie the associated inflammatory responses are poorly understood. In this context, we have studied the role played by Mitogen activated protein (MAP) kinase kinase 3 (MKK3), a dual-specificity protein kinase, in cigarette smoke induced-inflammation and mitochondrial dysfunction. Serum pro-inflammatory cytokines were significantly elevated in WT but not in MKK3-/- mice exposed to Cigarette smoke (CS) for 2 months. To study the cellular mechanisms of inflammation, bone marrow derived macrophages (BMDMs), wild type (WT) and MKK3-/-, were exposed to cigarette smoke extract (CSE) and inflammatory cytokine production and mitochondrial function assessed. The levels of IL-1ß, IL-6, and TNFα were increased along with higher reactive oxygen species (ROS) and P-NFκB after CSE treatment in WT but not in MKK3-/- BMDMs. CSE treatment adversely affected basal mitochondrial respiration, ATP production, maximum respiratory capacity, and spare respiratory capacity in WT BMDMs only. Mitophagy, clearance of dysfunctional mitochondria, was up regulated in CS exposed WT mice lung tissue and CSE exposed WT BMDMs, respectively. The proteomic analysis of BMDMs by iTRAQ (isobaric tags for relative and absolute quantitation) showed up regulation of mitochondrial dysfunction associated proteins in WT and higher OXPHOS (Oxidative phosphorylation) and IL-10 signaling proteins in MKK3-/- BMDMs after CSE exposure, confirming the critical role of mitochondrial homeostasis. Interestingly, we found increased levels of p-MKK3 by immunohistochemistry in COPD patient lung tissues that could be responsible for insufficient mitophagy and disease progression. This study identifies MKK3 as a negative regulator of mitochondrial function and inflammatory responses to CS and suggests that MKK3 could be a therapeutic target.

Inducing mitophagy in diabetic platelets protects against severe oxidative stress.

Diabetes mellitus (DM) is a growing international concern. Considerable mortality and morbidity associated with diabetes mellitus arise predominantly from thrombotic cardiovascular events. Oxidative stress-mediated mitochondrial damage contributes significantly to enhanced thrombosis in DM A basal autophagy process has recently been described as playing an important role in normal platelet activation. We now report a substantial mitophagy induction (above basal autophagy levels) in diabetic platelets, suggesting alternative roles for autophagy in platelet pathology. Using a combination of molecular, biochemical, and imaging studies on human DM platelets, we report that platelet mitophagy induction serves as a platelet protective mechanism that responds to oxidative stress through JNK activation. By removing damaged mitochondria (mitophagy), phosphorylated p53 is reduced, preventing progression to apoptosis, and preserving platelet function. The absence of mitophagy in DM platelets results in failure to protect against oxidative stress, leading to increased thrombosis. Surprisingly, this removal of damaged mitochondria does not require contributions from transcription, as platelets lack a nucleus. The considerable energy and resources expended in "prepackaging" the complex mitophagy machinery in a short-lived normal platelet support a critical role, in anticipation of exposure to oxidative stress.

Assessing hazardous risks of indoor airborne polycyclic aromatic hydrocarbons in the kitchen and its association with lung functions and urinary PAH metabolites in kitchen workers.

BACKGROUND: Indoor air pollution is associated with decreased pulmonary function but the relative impact of pollution from kitchen sources on health risks in kitchen workers is not well-known or studied. A study was conducted to measure the kitchen indoor air quality including PAHs estimation and risk assessment based on reported PAHs in indoor air in a central kitchen at North India. METHODS: A cross sectional study was undertaken to assess the lung function status using spirometer and urinary PAH metabolite measurements using GC-MS/MS among 94 male kitchen workers and their corresponding controls. Assessment of the indoor air quality levels was evaluated using standard methods. RESULTS: All the indoor air pollutants were within the recommended guidelines except CO, TVOC and PAH emission in the kitchen. Incremental life time cancer risk (ICLR) based on indoor air PAH measurements indicates potential for carcinogenic risk. Significant lung function decline was observed among kitchen workers as compared to controls after adjusting for smoking habits. Urinary PAH metabolites were detected in kitchen workers and measured concentrations were comparatively higher than control subjects. CONCLUSION: The decline in lung functions after adjustment for confounders and detection of urinary PAH metabolites in kitchen workers can be associated with higher concentrations of PAHs, CO and TVOCs in kitchen indoor air.

MKK3 deletion improves mitochondrial quality.

Sepsis, a severe response to infection, leads to excessive inflammation and is the major cause of mortality in intensive care units. Mitochondria have been shown to influence the outcome of septic injury. We have previously shown that MAP kinase kinase 3 (MKK3)(-/-) mice are resistant to septic injury and MKK3(-/-) macrophages have improved mitochondrial function. In this study we examined processes that lead to improved mitochondrial quality in MKK3(-/-) mouse embryonic fibroblasts (MEFs) and specifically the role of mitophagy in mitochondrial health. MKK3(-/-) MEFs had lower inflammatory cytokine release and oxidant production after lipopolysaccharide (LPS) stimulation, confirming our earlier observations. MKK3(-/-) MEFs had better mitochondrial function as measured by mitochondrial membrane potential (MMP) and ATP, even after LPS treatment. We observed higher mitophagy in MKK3(-/-) MEFs compared to wild type (WT). Transmission electron microscopy studies showed longer and larger mitochondria in MKK3(-/-) MEFs, indicative of healthier mitochondria. We performed a SILAC (stable isotope labeling by/with amino acids in cell culture) study to assess differences in mitochondrial proteome between WT and MKK3(-/-) MEFs and observed increased expression of tricarboxylic acid (TCA) cycle enzymes and respiratory complex subunits. Further, inhibition of mitophagy by Mdivi1 led to loss in MMP and increased cytokine secretion after LPS treatment in MKK3(-/-) MEFs. In conclusion, this study demonstrates that MKK3 influences mitochondrial quality by affecting the expression of mitochondrial proteins, including TCA cycle enzymes, and mitophagy, which consequently regulates the inflammatory response. Based on our results, MKK3 could be a potential therapeutic target for inflammatory diseases like sepsis.

Role of the bradykinin B2 receptor in a rat model of local heart irradiation.

PURPOSE: Radiation-induced heart disease (RIHD) is a delayed effect of radiotherapy for cancers of the chest, such as breast, esophageal, and lung. Kinins are small peptides with cardioprotective properties. We previously used a rat model that lacks the precursor kininogen to demonstrate that kinins are involved in RIHD. Here, we examined the role of the kinin B2 receptor (B2R) in early radiation-induced signaling in the heart. MATERIALS AND METHODS: Male Brown Norway rats received the B2R-selective antagonist HOE-140 (icatibant) via osmotic minipump from 5 days before until 4 weeks after 21 Gy local heart irradiation. At 4 weeks, signaling events were measured in left ventricular homogenates and nuclear extracts using western blotting and real-time polymerase chain reaction. Numbers of CD68-positive (monocytes/macrophages), CD2-positive (T-lymphocytes), and mast cells were measured using immunohistochemistry. RESULTS: Radiation-induced c-Jun phosphorylation and nuclear translocation were enhanced by HOE-140. HOE-140 did not modify endothelial nitric oxide synthase (eNOS) phosphorylation or alter numbers of CD2-positive or mast cells, but enhanced CD68-positive cell counts in irradiated hearts. CONCLUSIONS: B2R signaling may regulate monocyte/macrophage infiltration and c-Jun signals in the irradiated heart. Although eNOS is a main target for kinins, the B2R may not regulate eNOS phosphorylation in response to radiation.

MKK3 mediates inflammatory response through modulation of mitochondrial function.

Mitochondria are increasingly recognized as drivers of inflammatory responses. MAP kinase kinase 3 (MKK3), a dual-specificity protein kinase, is activated in inflammation and in turn activates p38 MAP kinase signaling. Here we show that MKK3 influences mitochondrial function and acts as a critical mediator of inflammation. MKK3-deficient (MKK3(-/-)) mice and bone marrow-derived macrophages (BMDMs) secreted smaller amounts of cytokines than wild type (WT) after lipopolysaccharide (LPS) exposure. There was improved mitochondrial function, as measured by basal oxygen consumption rate, mitochondrial membrane potential, and ATP production, in MKK3(-/-) BMDMs. After LPS exposure, MKK3(-/-) BMDMs did not show a significant increase in cellular reactive oxygen species production or in mitochondrial superoxide compared to WT. Activation of two important inflammatory mediators, i.e., the nuclear translocation of NF-κB and caspase-1 activity (a key component of the inflammasome), was lower in MKK3(-/-) BMDMs. p38 and JNK activation was lower in MKK3(-/-) BMDMs compared to WT after exposure to LPS. Knockdown of MKK3 by siRNA in wild-type BMDMs improved mitochondrial membrane potential, reduced LPS-induced caspase-1 activation, and attenuated cytokine secretion. Our studies establish MKK3 as a regulator of mitochondrial function and inflammatory responses to LPS and suggest that MKK3 may be a therapeutic target in inflammatory disorders such as sepsis.

Meta-analysis approach to study the prevalence of chronic obstructive pulmonary disease among current, former and non-smokers.

Comparative risk assessment for Chronic Obstructive Pulmonary Disease (COPD) among current, former and non-smokers categories remains controversial and not studied in detail. We conducted a meta-analysis to summarize all the relevant published studies on this topic and to update the association between smoking and prevalence of COPD in current, former and non-smokers. Identification, screening, eligibility and inclusion of articles for the study were conducted as per the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Quality assessment of included studies was undertaken using a scoring sheet. Meta-analysis after the final synthesis of the selected studies was performed using the STATA and Comprehensive Meta-Analysis (CMA) software. Estimates from forty two independent studies reporting 547,391 individuals were identified. Twenty two studies were conducted in Europe, nine in America and ten in Asia and one from New Zealand. The meta-analysis showed that the prevalence of COPD was significantly higher in current smokers compared with former and non-smokers. However, owing to large heterogeneity among the estimates obtained from the studies, stratification was done with respect to continent, diagnostic criteria of COPD and study design which also showed similar results. The stratified analysis also revealed similar trend of results with prevalence of COPD being higher in current smokers as compared to former and non-smokers. The present meta-analysis highlights the positive association between smoking and COPD prevalence. There is an urgent need to implement more effective policies towards the restriction of tobacco use, to reduce the burden of COPD.

MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury.

Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. Because specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Therefore targeted therapies to treat sepsis need to be developed. Although an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury as well as reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1, and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared with nonseptic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.