RESUMO
Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K+) solutions, we determined that CLIC6 is more permeable to chloride-(Cl-) as compared to bromide-(Br-), fluoride-(F-), and K+ ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 µM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl- channel.
Assuntos
Canais de Cloreto , Cloretos , Animais , Humanos , Camundongos , Ânions/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Células Epiteliais/metabolismo , Células HEK293RESUMO
Bronchopulmonary dysplasia (BPD) is a disease prevalent in preterm babies with a need for supplemental oxygen, resulting in impaired lung development and dysregulated vascularization. Epidemiologic studies have shown that males are more prone to BPD and have a delayed recovery compared with females, for reasons unknown. Herein, we tried to recapitulate mild, moderate, and severe BPD, using two different strains of mice, in males and females: CD1 (outbred) and C57BL/6 (inbred). Aside from higher body weight in the CD1 strain, there were no other gross morphologic differences with respect to alveolar development between the two strains. With respect to lung morphology after oxygen exposure, females had less injury with better preservation of alveolar chord length and decreased alveolar protein leak and inflammatory cells in the bronchoalveolar lavage fluid. In addition, housekeeping genes, which are routinely used as loading controls, were expressed differently in males and females. In the BPD mouse model, gonadotropin-releasing hormone was increased in females compared with males. Specific miRNAs (miR-146 and miR-34a) were expressed differently in the sexes. In the severe BPD mouse model, administering miR-146 mimic to males attenuated lung damage, whereas administering miR-146 inhibitor to females increased pulmonary injury.
Assuntos
Displasia Broncopulmonar/patologia , Modelos Animais de Doenças , Hiperóxia/complicações , Lesão Pulmonar/patologia , Índice de Gravidade de Doença , Animais , Displasia Broncopulmonar/etiologia , Feminino , Lesão Pulmonar/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores Sexuais , Especificidade da EspécieRESUMO
Mitochondria are the "power house" of a cell continuously generating ATP to ensure its proper functioning. The constant production of ATP via oxidative phosphorylation demands a large electrochemical force that drives protons across the highly selective and low-permeable mitochondrial inner membrane. Besides the conventional role of generating ATP, mitochondria also play an active role in calcium signaling, generation of reactive oxygen species (ROS), stress responses, and regulation of cell-death pathways. Deficiencies in these functions result in several pathological disorders like aging, cancer, diabetes, neurodegenerative and cardiovascular diseases. A plethora of ion channels and transporters are present in the mitochondrial inner and outer membranes which work in concert to preserve the ionic equilibrium of a cell for the maintenance of cell integrity, in physiological as well as pathophysiological conditions. For, e.g., mitochondrial cation channels KATP and BKCa play a significant role in cardioprotection from ischemia-reperfusion injury. In addition to the cation channels, mitochondrial anion channels are equally essential, as they aid in maintaining electro-neutrality by regulating the cell volume and pH. This chapter focusses on the information on molecular identity, structure, function, and physiological relevance of mitochondrial chloride channels such as voltage dependent anion channels (VDACs), uncharacterized mitochondrial inner membrane anion channels (IMACs), chloride intracellular channels (CLIC) and the aspects of forthcoming chloride channels.
Assuntos
Canais de Cloreto/fisiologia , Mitocôndrias/metabolismo , Canais de Ânion Dependentes de Voltagem/fisiologia , Animais , Humanos , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade MitocondrialRESUMO
BACKGROUND: Myocardial infarction (MI) is a significant cause of death in diabetic patients. Growing evidence suggests that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood. METHODS: We examined MRPL12 levels in right atrial appendage tissues from diabetic patients undergoing coronary artery bypass graft (CABG) surgery. Using AC-16 cells overexpressing MRPL12 under normal and hyperglycemic conditions we performed mitochondrial functional assays OXPHOS, bioenergetics, mitochondrial membrane potential, ATP production and cell death. RESULTS: We observed elevated MRPL12 levels in heart tissue samples from diabetic patients with ischemic heart disease compared to non-diabetic patients. Overexpression of MRPL12 under hyperglycemic conditions did not affect oxidative phosphorylation (OXPHOS) levels, cellular ATP levels, or cardiomyocyte cell death. However, notable impairment in mitochondrial membrane potential (MMP) was observed under hyperglycemic conditions, along with alterations in both basal respiration oxygen consumption rate (OCR) and maximal respiratory capacity OCR. CONCLUSIONS: Overall, our results suggest that MRPL12 may have a compensatory role in the diabetic myocardium with ischemic heart disease, suggesting that MRPL12 may implicate in the pathophysiology of MI in diabetes.
Assuntos
Proteínas de Ciclo Celular , Potencial da Membrana Mitocondrial , Isquemia Miocárdica , Proteínas Nucleares , Fosforilação Oxidativa , Proteínas Ribossômicas , Idoso , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Trifosfato de Adenosina/metabolismo , Apêndice Atrial/metabolismo , Apêndice Atrial/patologia , Ponte de Artéria Coronária , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/complicações , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Mitocôndrias Cardíacas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Isquemia Miocárdica/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Glycodelin A (GdA) is a dimeric glycoprotein synthesized by the human endometrium under progesterone regulation. Based on the high sequence similarity with ß-lactoglobulin, it is placed under the lipocalin superfamily. The protein is one of the local immunomodulators present at the feto-maternal interface which affects both the innate as well as the acquired arms of the immune system, thereby bringing about successful establishment and progression of pregnancy. Our previous studies revealed that the domain responsible for the immunosuppressive activity of glycodelin lies on its protein backbone and the glycans modulate the same. This study attempts to further delineate the apoptosis inducing region of GdA. Our results demonstrate that the stretch of amino acid sequence between Met24 to Leu105 is necessary and sufficient to inhibit proliferation of T cells and induce apoptosis in them. Further, within this region the key residues involved in harboring the activity were shown to be present between Asp52 and Ser65.
Assuntos
Apoptose , Glicoproteínas/fisiologia , Fatores Imunológicos/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Baculoviridae/genética , Proliferação de Células , Vetores Genéticos , Glicodelina , Glicoproteínas/química , Humanos , Fatores Imunológicos/química , Células Jurkat , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , Células Sf9 , Linfócitos T/fisiologiaRESUMO
OBJECTIVE: Despite great advances in obesity therapeutics in recent years, there is still a need to identify additional therapeutic targets for the treatment of this disease. We previously discovered a signature of genes, including Chloride intracellular channel 1 (Clic1), whose expression was associated with drug-induced weight gain, and in these studies, we assess the effect of Clic1 inhibition on food intake and body weight in mice. METHODS: We studied the impact of Clic1 inhibition in mouse models of binge-eating, diet-induced obese mice and genetic models of obesity (Magel2 KO mice). RESULTS: Clic1 knockout (KO) mice ate significantly less and had a lower body weight than WT littermates when either fed chow or high fat diet. Furthermore, pharmacological inhibition of Clic1 in diet-induced obese mice resulted in suppression of food intake and promoted highly efficacious weight loss. Clic1 inhibition also reduced food intake in binge-eating models and hyperphagic Magel2 KO mice. We observed that chronic obesity resulted in a significant change in subcellular localization of Clic1 with an increased ratio of Clic1 in the membrane in the obese state. These observations provide a novel therapeutic strategy to block Clic1 translocation as a potential mechanism to reduce food intake and lower body weight. CONCLUSIONS: These studies attribute a novel role of Clic1 as a driver of food intake and overconsumption. In summary, we have identified hypothalamic expression of Clic1 plays a key role in food intake, providing a novel therapeutic target to treat overconsumption that is the root cause of modern obesity.
Assuntos
Obesidade , Aumento de Peso , Animais , Camundongos , Camundongos Obesos , Peso Corporal , Camundongos Knockout , Ingestão de Alimentos , Canais de Cloreto/genética , Antígenos de Neoplasias , ProteínasRESUMO
BKCa channels are large-conductance calcium and voltage-activated potassium channels that are heterogeneously expressed in a wide array of cells. Activation of BKCa channels present in mitochondria of adult ventricular cardiomyocytes is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, the BKCa channel's activity has never been detected in the plasma membrane of adult ventricular cardiomyocytes. In this study, we report the presence of the BKCa channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes, which protects the heart on inhibition but not activation. Furthermore, K+ currents measured in neonatal cardiomyocyte (NCM) was sensitive to iberiotoxin (IbTx), suggesting the presence of BKCa channels in the plasma membrane. Neonatal hearts subjected to IR when post-conditioned with NS1619 during reoxygenation increased the myocardial infarction whereas IbTx reduced the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia and reoxygenation, whereas IbTx reduced TUNEL-positive cells. In NCMs, activation of BKCa channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BKCa channels in the NCM protects neonatal heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BKCa channels in adults compared to neonatal heart could be attributed to their differential localization.
RESUMO
Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.
RESUMO
BACKGROUND: It is difficult to simulate the abnormal myocardial strain patterns caused by ischemic coronary artery disease (CAD) which are a precursor to heart failure (HF) within an animal model. Simulation of these strain changes could contribute to better understanding of the early formative stages of HF. This is especially important in investigating the poorly understood pathogenesis of heart failure with preserved ejection fraction (HFpEF). Here, we discuss delivery of high intensity focused ultrasound (HIFU) in a murine model to alter left ventricular (LV) regional longitudinal strain (RLS), and use of speckle tracking echocardiography to detect these changes. METHODS: HIFU pulses (pressure amplitude 1.7 MPa) were generated by amplifying a sinusoidal waveform from a function generator into a piezoelectric transducer. These pulses were then directed extracorporeally towards the anterior LV surface of C57BI6 mice during three time periods (early, mid, and late diastole). Speckle tracking echocardiography was then used to quantify changes in RLS within six segments of the LV. RESULTS: We observed an increase in LV RLS with acoustic augmentation during all three time periods. This augmentation was most prominent near the anterior apical region in early diastole and near the posterior basilar region during late diastole. CONCLUSIONS: Our findings demonstrate the application of HIFU to non-invasively induce changes in RLS within a murine model. Our results also reflect the capability of speckle tracking echocardiography to analyze and quantify these changes. These findings represent the first demonstration of ultrasound-induced augmentation in LV RLS within a small animal model.
RESUMO
Cancer and heart diseases are the two leading causes of mortality and morbidity worldwide. Many cancer patients undergo heart-related complications resulting in high incidences of mortality. It is generally hypothesized that cardiac dysfunction in cancer patients occurs due to cardiotoxicity induced by therapeutic agents, used to treat cancers and/or cancer-induced cachexia. However, it is not known if localized tumors or unregulated cell growth systemically affect heart function before treatment, and/or prior to the onset of cachexia, hence, making the heart vulnerable to structural or functional abnormalities in later stages of the disease. We incorporated complementary mouse and Drosophila models to establish if tumor induction indeed causes cardiac defects even before intervention with chemotherapy or onset of cachexia. We focused on one of the key pathways involved in irregular cell growth, the Hippo-Yorkie (Yki), pathway. We used overexpression of the transcriptional co-activator of the Yki signaling pathway to induce cellular overgrowth, and show that Yki overexpression in the eye tissue of Drosophila results in compromised cardiac function. We rescue these cardiac phenotypes using antioxidant treatment, with which we conclude that the Yki induced tumorigenesis causes a systemic increase in ROS affecting cardiac function. Our results show that systemic cardiac dysfunction occurs due to abnormal cellular overgrowth or cancer elsewhere in the body; identification of specific cardiac defects associated with oncogenic pathways can facilitate the possible early diagnosis of cardiac dysfunction.
RESUMO
Mitochondria are the source of many pro-inflammatory signals that cause the activation of the immune system and generate inflammatory responses. They are also potential targets of pro-inflammatory mediators, thus triggering a severe inflammatory response cycle. As mitochondria are a central hub for immune system activation, their dysfunction leads to many inflammatory disorders. Thus, strategies aiming at regulating mitochondrial dysfunction can be utilized as a therapeutic tool to cure inflammatory disorders. Two key factors that determine the structural and functional integrity of mitochondria are mitochondrial ion channels and transporters. They are not only important for maintaining the ionic homeostasis of the cell, but also play a role in regulating reactive oxygen species generation, ATP production, calcium homeostasis and apoptosis, which are common pro-inflammatory signals. The significance of the mitochondrial ion channels in inflammatory response is still not clearly understood and will need further investigation. In this article, we review the different mechanisms by which mitochondria can generate the inflammatory response as well as highlight how mitochondrial ion channels modulate these mechanisms and impact the inflammatory processes.
RESUMO
OBJECTIVE: To quantify and compare levels of potential biomarkers in neonates with (i) Bronchopulmonary dysplasia (BPD); (ii) BPD-associated pulmonary hypertension (BPD-PH); (iii) PH without BPD; and (iv) neonates without lung disease at ~36 weeks postmenstrual age. STUDY DESIGN: Multiple potential biomarkers were measured in plasma samples of 90 patients using a multi-spot enzyme-linked immunosorbent assay. Statistical tests done included one-way ANOVA to compare levels of biomarkers between different groups. RESULTS: Higher levels of ICAM-1 were present in infants with BPD and correlated with its severity. Infants with BPD have significantly higher levels of ANG-2 and lower levels of ANG-1. Infants with PH have higher levels of: IL-6, IL-8, IL-10, and TNF-α. Infants with BPD-PH have significantly lower levels of MCP-1 and higher levels of IL-1ß than infants with PH without BPD. CONCLUSION: ICAM-1 may be used as a specific biomarker for diagnosis of BPD and its severity.
Assuntos
Displasia Broncopulmonar , Hipertensão Pulmonar , Biomarcadores/análise , Displasia Broncopulmonar/diagnóstico , Feminino , Humanos , Hipertensão Pulmonar/diagnóstico , Lactente , Recém-Nascido , GravidezRESUMO
Among the many nonviral gene delivery vectors, chitosan, being a polysaccharide of natural origin, has gained special importance. In this report, chitosan (CS) has been solubilized in water by preparing its O-carboxymethyl derivative, CS(CH2COOH), with an optimum degree of carboxymethylation. This has been further derivatized to get the pyridine-substituted product (py)CS(CH2COOH), where the degree of pyridine substitution (47%) was optimized based on zeta potential measurements. The optimized formulation showed a high gene binding ability, forming nanosized positively charged polyelectrolyte complexes with DNA. These polyplexes were stable to DNase and physiological polyanions such as heparin. They also exhibited minimal toxicity in vitro and showed transfection levels comparable to the commercial standard Lipofectamine 2000 and much higher than polyethylenimine (MW, 25 kDa). Additionally, in this study, a hitherto unknown oxyamine derivative of chitosan has been prepared by phthaloyl protection, tosylation, and Gabriel's phthalimide synthesis. Nearly 40% of the primary alcohols were successfully converted to oxyamino functionality, which was used for forming oxime with the anticancer drug doxorubicin. The pH sensitivity of the oxime ether linkage and stability under biologically relevant conditions were then used to establish the compound as a versatile drug delivery vector. Co-delivery of functional gene (p53) and drug (doxorubicin) was accomplished in vitro and in vivo with the chitosan-pyridine imine vector (py)CS(CH2COOH) and the newly synthesized doxorubicin oxime ether CS(Dox). Complete tumor regression with no tumor recurrence and appreciable survivability point to the in vivo effectiveness and biocompatibility of the designed composite formulation. Overall, the pH sensitivity of the oxime linkage aiding slow and steady drug release, together with the sustained gene expression by pyridine-tethered carboxymethyl chitosan, allows us to generate a nanobiocomposite with significantly high anticancer therapeutic potential.
Assuntos
Quitosana , Doxorrubicina , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Terapia Genética , Neoplasias Experimentais , Proteína Supressora de Tumor p53 , Animais , Linhagem Celular Tumoral , Quitosana/análogos & derivados , Quitosana/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Solubilidade , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
Indanyloxyacetic acid-94 (IAA-94), an intracellular chloride channel blocker, is shown to ablate cardioprotection rendered by ischemic preconditioning (IPC), N (6)-2-(4-aminophenyl) ethyladenosine or the PKC activator phorbol 12-myristate 13-acetate and cyclosporin A (CsA) in both ex-vivo and in-vivo ischemia-reperfusion (IR) injury. Thus signifying the role of the IAA-94 sensitive chloride channels in mediating cardio-protection upon IR injury. Although IAA-94 sensitive chloride currents are recorded in cardiac mitoplast, there is still a lack of understanding of the mechanism by which IAA-94 increases myocardial infarction (MI) by IR injury. Mitochondria are the key arbitrators of cell life and death pathways. Both oxidative stress and calcium overload in the mitochondria, elicit pathways resulting in the opening of mitochondrial permeability transition pore (mPTP) leading to cell death. Therefore, in this study we explored the role of IAA-94 in MI and in maintaining calcium retention capacity (CRC) of cardiac mitochondria after IR. IAA-94 inhibited the CRC of the isolated cardiac mitochondria in a concentration-dependent manner as measured spectrofluorimetrically using calcium green-5â¯N. Interestingly, IAA-94 did not change the mitochondrial membrane potential. Further, CsA a blocker of mPTP opening could not override the effect of IAA-94. We also showed for the first time that IAA-94 perfusion after ischemic event augments MI by reducing the CRC of mitochondria. To conclude, our results demonstrate that the mechanism of IAA-94 mediated cardio-deleterious effects is via modulating the mitochondria CRC, thereby playing a role in mPTP opening. These findings highlight new pharmacological targets, which can mediate cardioprotection from IR injury.
Assuntos
Cálcio/metabolismo , Glicolatos/efeitos adversos , Infarto do Miocárdio/metabolismo , Animais , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Glicolatos/antagonistas & inibidores , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Infarto do Miocárdio/induzido quimicamente , RatosRESUMO
BKCa channels, originally discovered in Drosophila melanogaster as slowpoke (slo), are recognized for their roles in cellular and organ physiology. Pharmacological approaches implicated BKCa channels in cellular and organ protection possibly for their ability to modulate mitochondrial function. However, the direct role of BKCa channels in regulating mitochondrial structure and function is not deciphered. Here, we demonstrate that BKCa channels are present in fly mitochondria, and slo mutants show structural and functional defects in mitochondria. slo mutants display an increase in reactive oxygen species and the modulation of ROS affected their survival. We also found that the absence of BKCa channels reduced the lifespan of Drosophila, and overexpression of human BKCa channels in flies extends life span in males. Our study establishes the presence of BKCa channels in mitochondria of Drosophila and ascertains its novel physiological role in regulating mitochondrial structural and functional integrity, and lifespan.
Assuntos
Drosophila melanogaster/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Longevidade , Mitocôndrias/metabolismo , AnimaisRESUMO
Intracellular organelles are membranous structures central for maintaining cellular physiology and the overall health of the cell. To maintain cellular function, intracellular organelles are required to tightly regulate their ionic homeostasis. Any imbalance in ionic concentrations can disrupt energy production (mitochondria), protein degradation (lysosomes), DNA replication (nucleus), or cellular signaling (endoplasmic reticulum). Ionic homeostasis is also important for volume regulation of intracellular organelles and is maintained by cation and anion channels as well as transporters. One of the major classes of ion channels predominantly localized to intracellular membranes is chloride intracellular channel proteins (CLICs). They are non-canonical ion channels with six homologs in mammals, existing as either soluble or integral membrane protein forms, with dual functions as enzymes and channels. Provided in this overview is a brief introduction to CLICs, and a summary of recent information on their localization, biophysical properties, and physiological roles. © 2018 by John Wiley & Sons, Inc.
Assuntos
Canais de Cloreto/fisiologia , Animais , Agonistas dos Canais de Cloreto , Canais de Cloreto/antagonistas & inibidores , Humanos , OrganelasRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) are promising tools for the treatment of human lung disease and other pathologies relevant to newborn medicine. Recent studies have established MSC exosomes (EXO), as one of the main therapeutic vectors of MSCs in mouse models of multifactorial chronic lung disease of preterm infants, bronchopulmonary dysplasia (BPD). However, the mechanisms underlying MSC-EXO therapeutic action are not completely understood. Using a neonatal mouse model of human BPD, we evaluated the therapeutic efficiency of early gestational age (GA) human umbilical cord (hUC)-derived MSC EXO fraction and its exosomal factor, tumor necrosis factor alpha-stimulated gene-6 (TSG-6). METHODS: Conditioned media (CM) and EXO fractions were isolated from 25 and 30 weeks GA hUC-MSC cultures grown in serum-free media (SFM) for 24 h. Newborn mice were exposed to hyperoxia (> 95% oxygen) and were given intraperitoneal injections of MSC-CM or MSC-CM EXO fractions at postnatal (PN) day 2 and PN4. They were then returned to room air until PN14 (in a mouse model of severe BPD). The treatment regime was followed with (rh)TSG-6, TSG-6-neutralizing antibody (NAb), TSG-6 (si)RNA-transfected MSC-CM EXO and their appropriate controls. Echocardiography was done at PN14 followed by harvesting of lung, heart and brain for assessment of pathology parameters. RESULTS: Systemic administration of CM or EXO in the neonatal BPD mouse model resulted in robust improvement in lung, cardiac and brain pathology. Hyperoxia-exposed BPD mice exhibited pulmonary inflammation accompanied by alveolar-capillary leakage, increased chord length, and alveolar simplification, which was ameliorated by MSC CM/EXO treatment. Pulmonary hypertension and right ventricular hypertrophy was also corrected. Cell death in brain was decreased and the hypomyelination reversed. Importantly, we detected TSG-6, an immunomodulatory glycoprotein, in EXO. Administration of TSG-6 attenuated BPD and its associated pathologies, in lung, heart and brain. Knockdown of TSG-6 by NAb or by siRNA in EXO abrogated the therapeutic effects of EXO, suggesting TSG-6 as an important therapeutic molecule. CONCLUSIONS: Preterm hUC-derived MSC-CM EXO alleviates hyperoxia-induced BPD and its associated pathologies, in part, via exosomal factor TSG-6. The work indicates early systemic intervention with TSG-6 as a robust option for cell-free therapy, particularly for treating BPD.
Assuntos
Displasia Broncopulmonar/genética , Moléculas de Adesão Celular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/patologia , Modelos Animais de Doenças , Humanos , Recém-Nascido , CamundongosRESUMO
Aims: Activation and expression of large conductance calcium and voltage-activated potassium channel (BKCa) by pharmacological agents have been implicated in cardioprotection from ischemia-reperfusion (IR) injury possibly by regulating mitochondrial function. Given the non-specific effects of pharmacological agents, it is not clear whether activation of BKCa is critical to cardioprotection. In this study, we aimed to decipher the mechanistic role of BKCa in cardioprotection from IR injury by genetically activating BKCa channels. Methods and Results: Hearts from adult (3 months old) wild-type mice (C57/BL6) and mice expressing genetically activated BKCa (Tg-BKCa R207Q, referred as Tg-BKCa) along with wild-type BKCa were subjected to 20 min of ischemia and 30 min of reperfusion with or without ischemic preconditioning (IPC, 2 times for 2.5 min interval each). Left ventricular developed pressure (LVDP) was recorded using Millar's Mikrotip® catheter connected to ADInstrument data acquisition system. Myocardial infarction was quantified by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Our results demonstrated that Tg-BKCa mice are protected from IR injury, and BKCa also contributes to IPC-mediated cardioprotection. Cardiac function parameters were also measured by echocardiography and no differences were observed in left ventricular ejection fraction, fractional shortening and aortic velocities. Amplex Red® was used to assess reactive oxygen species (ROS) production in isolated mitochondria by spectrofluorometry. We found that genetic activation of BKCa reduces ROS after IR stress. Adult cardiomyocytes and mitochondria from Tg-BKCa mice were isolated and labeled with Anti-BKCa antibodies. Images acquired via confocal microscopy revealed localization of cardiac BKCa in the mitochondria. Conclusions: Activation of BKCa is essential for recovery of cardiac function after IR injury and is likely a factor in IPC mediated cardioprotection. Genetic activation of BKCa reduces ROS produced by complex I and complex II/III in Tg-BKCa mice after IR, and IPC further decreases it. These results implicate BKCa-mediated cardioprotection, in part, by reducing mitochondrial ROS production. Localization of Tg-BKCa in adult cardiomyocytes of transgenic mice was similar to BKCa in wild-type mice.
RESUMO
Large conductance calcium and voltage-activated potassium channels (BKCa ) are transmembrane proteins, ubiquitously expressed in the majority of organs, and play an active role in regulating cellular physiology. In the heart, BKCa channels are known to play a role in regulating the heart rate and protect it from ischemia-reperfusion injury. In vascular smooth muscle cells, the opening of BKCa channels results in membrane hyperpolarization which eventually results in vasodilation mediated by a reduction in Ca2+ influx due to the closure of voltage-dependent Ca2+ channels. Ex vivo studies have shown that BKCa channels play an active role in the regulation of the function of the majority of blood vessels. However, in vivo role of BKCa channels in cardiovascular function is not completely deciphered. Here, we have evaluated the rapid in vivo role of BKCa channels in regulating the cardiovascular function by using two well-established, rapid-acting, potent blockers, paxilline and iberiotoxin. Our results show that BKCa channels are actively involved in regulating the heart rate, the function of the left and right heart as well as major vessels. We also found that the effect on BKCa channels by blockers is completely reversible, and hence, BKCa channels can be exploited as potential targets for clinical applications for modulating heart rate and cardiac contractility.
Assuntos
Frequência Cardíaca/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Função Ventricular/fisiologia , Animais , Velocidade do Fluxo Sanguíneo/fisiologia , Circulação Coronária/efeitos dos fármacos , Circulação Coronária/fisiologia , Ecocardiografia , Coração/diagnóstico por imagem , Frequência Cardíaca/efeitos dos fármacos , Indóis/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Artéria Pulmonar/diagnóstico por imagem , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Ratos Sprague-Dawley , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular/efeitos dos fármacosRESUMO
Chloride intracellular channels (CLIC) are non-classical ion channels lacking a signal sequence for membrane targeting. In eukaryotes, they are implicated in cell volume regulation, acidification, and cell cycle. CLICs resemble the omega class of Glutathione S-transferases (GST), yet differ from them in their ability to form ion channels. They are ubiquitously found in eukaryotes but no prokaryotic homolog has been characterized. We found that indanyloxyacetic acid-94 (IAA-94), a blocker of CLICs, delays the growth of Escherichia coli. In silico analysis showed that the E. coli stringent starvation protein A (SspA) shares sequence and structural homology with CLICs. Similar to CLICs, SspA lacks a signal sequence but contains an omega GST fold. Electrophysiological analysis revealed that SspA auto-inserts into lipid bilayers and forms IAA-94-sensitive ion channels. Substituting the ubiquitously conserved residue leucine 29 to alanine in the pore-forming region increased its single-channel conductance. SspA is essential for cell survival during acid-induced stress, and we found that acidic pH increases the open probability of SspA. Further, IAA-94 delayed the growth of wild-type but not sspA null mutant E. coli. Our results for the first time show that CLIC-like proteins exist in bacteria in the form of SspA, forming functional ion channels.