Aberrant PGC-1α signaling in a lamb model of persistent pulmonary hypertension of the newborn.

BACKGROUND: Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by elevated pulmonary vascular resistance (PVR), resulting in hypoxemia. Impaired angiogenesis contributes to high PVR. Pulmonary artery endothelial cells (PAECs) in PPHN exhibit decreased mitochondrial respiration and angiogenesis. We hypothesize that Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α) downregulation leads to reduced mitochondrial function and angiogenesis in PPHN. METHODS: Studies were performed in PAECs isolated from fetal lambs with PPHN induced by ductus arteriosus constriction, with gestation-matched controls and in normal human umbilical vein endothelial cells (HUVECs). PGC-1α was knocked downed in control lamb PAECs and HUVECs and overexpressed in PPHN PAECs to investigate the effects on mitochondrial function and angiogenesis. RESULTS: PPHN PAECs had decreased PGC-1α expression compared to controls. PGC-1α knockdown in HUVECs led to reduced Nuclear Respiratory Factor-1 (NRF-1), Transcription Factor-A of Mitochondria (TFAM), and mitochondrial electron transport chain (ETC) complexes expression. PGC-1α knockdown in control PAECs led to decreased in vitro capillary tube formation, cell migration, and proliferation. PGC-1α upregulation in PPHN PAECs led to increased ETC complexes expression and improved tube formation, cell migration, and proliferation. CONCLUSION: PGC-1α downregulation contributes to reduced mitochondrial oxidative phosphorylation through control of the ETC complexes, thereby affecting angiogenesis in PPHN. IMPACT: Reveals a novel mechanism for angiogenesis dysfunction in persistent pulmonary hypertension of the newborn (PPHN). Identifies a key mitochondrial transcription factor, Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α), as contributing to the altered adaptation and impaired angiogenesis function that characterizes PPHN through its regulation of mitochondrial function and oxidative phosphorylation. May provide translational significance as this mechanism offers a new therapeutic target in PPHN, and efforts to restore PGC-1α expression may improve postnatal transition in PPHN.

Decreased Liver Kinase B1 Expression and Impaired Angiogenesis in a Murine Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is characterized by impaired lung alveolar and vascular growth. We investigated the hypothesis that neonatal exposure to hyperoxia leads to persistent BPD phenotype caused by decreased expression of liver kinase B1 (LKB1), a key regulator of mitochondrial function. We exposed mouse pups from Postnatal Day (P)1 through P10 to 21% or 75% oxygen. Half of the pups in each group received metformin or saline intraperitoneally from P1 to P10. Pups were killed at P4 or P10 or recovered in 21% O2 until euthanasia at P21. Lung histology and morphometry, immunofluorescence, and immunoblots were performed to detect changes in lung structure and expression of LKB1; downstream targets AMPK, PGC-1α, and electron transport chain (ETC) complexes; and Notch ligands Jagged 1 and delta-like 4. LKB1 signaling and in vitro angiogenesis were assessed in human pulmonary artery endothelial cells (exposed to 21% or 95% O2 for 36 hours. Levels of LKB1, phosphorylated AMPK, PGC-1α, and ETC complexes were decreased in lungs at P10 and P21 in hyperoxia. Metformin increased LKB1, phosphorylated AMPK, PGC-1α, and ETC complexes at P10 and P21 in pups exposed to hyperoxia. Radial alveolar count was decreased, and mean linear intercept increased in pups exposed to hyperoxia at P10 and P21; these were improved by metformin. Lung capillary density was decreased in hyperoxia at P10 and P21 and was increased by metformin. In vitro angiogenesis was decreased in human pulmonary artery endothelial cells by 95% O2 and was improved by metformin. Decreased LKB1 signaling may contribute to decreased alveolar and vascular growth in a mouse model of BPD.

Fetal pulmonary hypertension: dysregulated microRNA-34c-Notch1 axis contributes to impaired angiogenesis in an ovine model.

BACKGROUND: Persistent pulmonary hypertension of the newborn (PPHN) occurs when pulmonary vascular resistance (PVR) fails to decrease at birth. Decreased angiogenesis in the lung contributes to the persistence of high PVR at birth. MicroRNAs (miRNAs) regulate gene expression through transcript binding and degradation. They were implicated in dysregulated angiogenesis in cancer and cardiovascular disease. METHODS: We investigated whether altered miRNA levels contribute to impaired angiogenesis in PPHN. We used a fetal lamb model of PPHN induced by prenatal ductus arteriosus constriction and sham ligation as controls. We performed RNA sequencing of pulmonary artery endothelial cells (PAECs) isolated from control and PPHN lambs. RESULTS: We observed a differentially expressed miRNA profile in PPHN for organ development, cell-cell signaling, and cardiovascular function. MiR-34c was upregulated in PPHN PAECs compared to controls. Exogenous miR34c mimics decreased angiogenesis by control PAEC and anti-miR34c improved angiogenesis of PPHN PAEC in vitro. Notch1, a predicted target for miR-34c by bioinformatics, was decreased in PPHN PAECs, along with Notch1 downstream targets, Hey1 and Hes1. Exogenous miR-34c decreased Notch1 expression in control PAECs and anti-miR-34c restored Notch1 and Hes1 expression in PPHN PAECs. CONCLUSION: We conclude that increased miR-34c in PPHN contributes to impaired angiogenesis by decreasing Notch1 expression in PAECs. IMPACT: Adds a novel mechanism for the regulation of angiogenesis in persistent pulmonary hypertension of the newborn. Identifies non-coding RNAs that are involved in the altered angiogenesis in PPHN and thus the potential for future studies to identify links between known pathways regulating angiogenesis. Provides preliminary data to conduct studies targeting miR34c expression in vivo in animal models of pulmonary hypertension to identify the mechanistic role of miR34c in angiogenesis in the lung vasculature.

NOGOB receptor deficiency increases cerebrovascular permeability and hemorrhage via impairing histone acetylation-mediated CCM1/2 expression.

The loss function of cerebral cavernous malformation (CCM) genes leads to most CCM lesions characterized by enlarged leaking vascular lesions in the brain. Although we previously showed that NOGOB receptor (NGBR) knockout in endothelial cells (ECs) results in cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NGBR regulates CCM1/2 expression has not been elucidated. Here, we show that genetic depletion of Ngbr in ECs at both postnatal and adult stages results in CCM1/2 expression deficiency and cerebrovascular lesions such as enlarged vessels, blood-brain-barrier hyperpermeability, and cerebral hemorrhage. To reveal the molecular mechanism, we used RNA-sequencing analysis to examine changes in the transcriptome. Surprisingly, we found that the acetyltransferase HBO1 and histone acetylation were downregulated in NGBR-deficient ECs. The mechanistic studies elucidated that NGBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NGBR impairs the binding of HBO1 and acetylated histone H4K5 and H4K12 on the promotor of the CCM1 and CCM2 genes. Our findings on epigenetic regulation of CCM1 and CCM2 that is modulated by NGBR and HBO1-mediated histone H4 acetylation provide a perspective on the pathogenesis of sporadic CCMs.

Decreased Cyclic Guanosine Monophosphate-Protein Kinase G Signaling Impairs Angiogenesis in a Lamb Model of Persistent Pulmonary Hypertension of the Newborn.

Impaired angiogenesis function in pulmonary artery endothelial cells (PAEC) contributes to persistent pulmonary hypertension of the newborn (PPHN). Decreased nitric oxide (NO) amounts in PPHN lead to impaired mitochondrial biogenesis and angiogenesis in the lung; the mechanisms remain unclear. We hypothesized that decreased cyclic guanosine monophosphate (cGMP)-PKG (protein kinase G) signaling downstream of NO leads to decreased mitochondrial biogenesis and angiogenesis in PPHN. PPHN was induced by ductus arteriosus constriction from 128-136 days' gestation in fetal lambs. Control animals were gestation-matched lambs that did not undergo ductal constriction. PAEC isolated from PPHN lambs were treated with the sGC (soluble guanylate cyclase) activator cinaciguat, the PKG activator 8-bromo-cGMP, or the PDE-V (PDE type V) inhibitor sildenafil. Lysates were immunoblotted for mitochondrial transcription factors and electron transport chain C-I (complex I), C-II, C-III, C-IV, and C-V proteins. The in vitro angiogenesis of PAEC was evaluated by using tube-formation and scratch-recovery assays. cGMP concentrations were measured by using an enzyme immunoassay. Fetal lambs with ductal constriction were given sildenafil or control saline through continuous infusion in utero, and the lung histology, capillary counts, vessel density, and right ventricular pressure were assessed at birth. PPHN PAEC showed decreased mitochondrial transcription factor levels, electron transport chain protein levels, and in vitro tube formation and cell migration; these were restored by cinaciguat, 8-bromo-cGMP, and sildenafil. Cinaciguat and sildenafil increased cGMP concentrations in PPHN PAEC. Radial alveolar and capillary counts and vessel density were lower in PPHN lungs, and the right ventricular pressure and Fulton Index were higher in PPHN lungs; these were improved by in utero sildenafil infusion. cGMP-PKG signaling is a potential therapeutic target to restore decreased mitochondrial biogenesis and angiogenesis in PPHN.

Decreased AMP-activated protein kinase (AMPK) function and protective effect of metformin in neonatal rat pups exposed to hyperoxia lung injury.

We investigated the hypothesis that exposure of lungs at the saccular stage of development to hyperoxia leads to persistent growth arrest and dysfunction of 5'AMP-activated protein kinase (AMPK), a key energy sensor in the cell. We exposed neonatal rat pups from postnatal day 1- day 10 (P1-P10) to ≥90% oxygen or control normoxia. Pups were euthanized at P4 or P10 or recovered in normoxia until euthanasia at P21. Half of the pups in each group received AMPK activator, metformin, or saline intraperitoneally from P1 to P10. Lung histology, morphometric analysis, immunofluorescence, and immunoblots were done for changes in lung structure at P10 and P21 and AMPK function at P4, P10, and P21. Phosphorylation of AMPK (p-AMPK) was decreased in lungs at P10 and P21 in hyperoxia-exposed pups. Metformin increased the levels of p-AMPK and PGC-1α, a downstream AMPK target which regulates mitochondrial biogenesis, at P4, P10, and P21 in hyperoxia pups. Lung ATP levels decreased during hyperoxia and were increased by metformin at P10 and P21. Radial alveolar count and alveolar septal tips were decreased and mean linear intercept increased in hyperoxia-exposed pups at P10 and the changes persisted at P21; these were improved by metformin. Lung capillary number was decreased in hyperoxia-exposed pups at P10 and P21 and was restored by metformin. Hyperoxia leads to impaired AMPK function, energy balance and alveolar simplification. The AMPK activator, metformin improves AMPK function and alveolar and vascular growth in this rat pup model of hyperoxia-induced lung injury.

AMP-Kinase Dysfunction Alters Notch Ligands to Impair Angiogenesis in Neonatal Pulmonary Hypertension.

Decreased angiogenesis contributes to persistent pulmonary hypertension of the newborn (PPHN); mechanisms remain unclear. AMPK (5'AMP activated protein kinase) is a key regulator of cell metabolism. We investigated the hypothesis that a decrease in AMPK function leads to mitochondrial dysfunction and altered balance of notch ligands delta-like 4 (DLL4) and Jagged 1 (Jag1) to impair angiogenesis in PPHN. Studies were done in fetal lambs with PPHN induced by prenatal ductus arteriosus constriction and gestation-matched control lambs. PPHN lambs were treated with saline or AMPK agonist metformin. Angiogenesis was assessed in lungs with micro-computed tomography angiography and histology. AMPK function; expression of mitochondrial electron transport chain (ETC) complex proteins I-V, Dll4, and Jag1; mitochondrial number; and in vitro angiogenesis function were assessed in pulmonary artery endothelial cells (PAEC) from control and PPHN lambs. AMPK function was decreased in PPHN PAEC and lung sections. Expression of mitochondrial transcription factor, PGC-1α, ETC complex proteins I-V, and mitochondrial number were decreased in PPHN. In vitro angiogenesis of PAEC and capillary number and vessel volume fraction in the lung were decreased in PPHN. Expression of DLL4 was increased and Jag1 was decreased in PAEC from PPHN lambs. AMPK agonists A769662 and metformin increased the mitochondrial complex proteins and number, in vitro angiogenesis, and Jag1 levels and decreased DLL4 levels in PPHN PAEC. Infusion of metformin in vivo increased the vessel density in PPHN lungs. Decreased AMPK function contributes to impaired angiogenesis in PPHN by altered balance of notch ligands in PPHN.

Adherence to endocrine therapy among Chinese patients with breast cancer: current status and recommendations for improvement.

BACKGROUND: Postoperative endocrine therapy is known to reduce recurrence and mortality in patients with estrogen receptor (ER)- or progestogen receptor (PR)-positive breast cancer. Correlates and determinants of compliance with endocrine therapy among Chinese patients with breast cancer are not known. The aim of this study was to elucidate the efficacy and adherence of endocrine therapy in China and suggest effective improvements on the adherence. PATIENTS AND METHODS: We analyzed the survival of 1,110 patients eligible for endocrine therapy and adherence of 699 patients to endocrine therapy. Kaplan-Meier curves, log-rank tests and Cox proportional hazard models were used to evaluate survival, and logistic regression models were used to assess variables associated with treatment adherence. RESULTS: Long-term endocrine therapy was associated with lower recurrence rate (HR 0.72; 95% CI 0.56-0.93; p=0.013). Adherence to endocrine therapy was only 63.1%. Sociodemographic characteristics of patients, clinical- and medication-related characteristics and patients' attitudes were associated with adherence to endocrine therapy. CONCLUSION: Adherence to endocrine therapy in Chinese patients with ER+/PR+ breast cancer was <65%. Both patients and physicians should take progressive steps to improve the rate of adherence.

Nogo-B receptor increases the resistance of estrogen receptor positive breast cancer to paclitaxel.

Intrinsic or acquired chemoresistance is a hurdle in oncology. Only 7%-16% of estrogen receptor α (ERα) positive breast cancer cases achieve a pathological complete response (pCR) after neo-adjuvant chemotherapy. Nogo-B receptor (NgBR) is a cell surface receptor that binds farnesylated Ras and promotes Ras translocation to the plasma membrane. Here, we demonstrate NgBR as a potential therapeutic target for ERα positive breast cancer patients to attenuate paclitaxel resistance. NgBR knockdown enhanced paclitaxel-induced cell apoptosis by modulating expression of p53 and survivin in ERα positive breast cancer cells via NgBR-mediated PI3K/Akt and MAPK/ERK signaling pathways. NgBR knockdown attenuated either 17ß-estradiol or epidermal growth factor stimulated phosphorylation of ERα at Serine 118 residue. The ChIP-PCR assay further demonstrated that NgBR knockdown decreased ERα binding to the estrogen response element (ERE) of the ERα target gene and increased the binding of p53 to the promoter region of survivin to attenuate survivin transcription. In summary, our data suggest that NgBR expression is essential to promoting ERα positive breast cancer cell resistance to paclitaxel. Findings from this study implicate a novel therapeutic target for treating ERα positive breast cancer in neo-adjuvant/adjuvant chemotherapy.

Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function.

Stress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at 445GERAMT450 Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity in vitro and in vivo To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu446 and Arg447 cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.

Nogo-B receptor deficiency increases liver X receptor alpha nuclear translocation and hepatic lipogenesis through an adenosine monophosphate-activated protein kinase alpha-dependent pathway.

Nogo-B receptor (NgBR) was identified as a specific receptor for binding Nogo-B and is essential for the stability of Niemann-Pick type C2 protein (NPC2) and NPC2-dependent cholesterol trafficking. Here, we report that NgBR expression levels decrease in the fatty liver and that NgBR plays previously unrecognized roles in regulating hepatic lipogenesis through NPC2-independent pathways. To further elucidate the pathophysiological role of NgBR in mammals, we generated NgBR liver-specific knockout mice and investigated the roles of NgBR in hepatic lipid homeostasis. The results showed that NgBR knockout in mouse liver did not decrease NPC2 levels or increase NPC2-dependent intracellular cholesterol levels. However, NgBR deficiency still resulted in remarkable cellular lipid accumulation that was associated with increased free fatty acids and triglycerides in hepatocytes in vitro and in mouse livers in vivo. Mechanistically, NgBR deficiency specifically promotes the nuclear translocation of the liver X receptor alpha (LXRα) and increases the expression of LXRα-targeted lipogenic genes. LXRα knockout attenuates the accumulation of free fatty acids and triglycerides caused by NgBR deficiency. In addition, we elucidated the mechanisms by which NgBR bridges the adenosine monophosphate-activated protein kinase alpha signaling pathway with LXRα nuclear translocation and LXRα-mediated lipogenesis. CONCLUSION: NgBR is a specific negative regulator for LXRα-dependent hepatic lipogenesis. Loss of NgBR may be a potential trigger for inducing hepatic steatosis. (Hepatology 2016;64:1559-1576).

Nogo-B receptor deficiency causes cerebral vasculature defects during embryonic development in mice.

Nogo-B receptor (NgBR) was identified as a receptor specific for Nogo-B. Our previous work has shown that Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and intersomitic vessel formation via Akt pathway in zebrafish. Here, we further demonstrated the roles of NgBR in regulating vasculature development in mouse embryo and primitive blood vessel formation in embryoid body culture systems, respectively. Our results showed that NgBR homozygous knockout mice are embryonically lethal at E7.5 or earlier, and Tie2Cre-mediated endothelial cell-specific NgBR knockout (NgBR ecKO) mice die at E11.5 and have severe blood vessel assembly defects in embryo. In addition, mutant embryos exhibit dilation of cerebral blood vessel, resulting in thin-walled endothelial caverns. The similar vascular defects also were detected in Cdh5(PAC)-CreERT2 NgBR inducible ecKO mice. Murine NgBR gene-targeting embryonic stem cells (ESC) were generated by homologous recombination approaches. Homozygous knockout of NgBR in ESC results in cell apoptosis. Heterozygous knockout of NgBR does not affect ESC cell survival, but reduces the formation and branching of primitive blood vessels in embryoid body culture systems. Mechanistically, NgBR knockdown not only decreases both Nogo-B and VEGF-stimulated endothelial cell migration by abolishing Akt phosphorylation, but also decreases the expression of CCM1 and CCM2 proteins. Furthermore, we performed immunofluorescence (IF) staining of NgBR in human cerebral cavernous malformation patient tissue sections. The quantitative analysis results showed that NgBR expression levels in CD31 positive endothelial cells is significantly decreased in patient tissue sections. These results suggest that NgBR may be one of important genes coordinating the cerebral vasculature development.

Nogo-B Receptor Modulates Pulmonary Artery Smooth Muscle Cell Function in Developing Lungs.

Nogo-B and its receptor (NgBR) are involved in blood vessel growth in developing lungs, but their role in pulmonary artery smooth muscle cell (PASMC) growth is unknown. We hypothesized that NgBR regulates growth of PASMCs by modulating the function of endoplasmic reticulum (ER) and formation of reactive oxygen species (ROS). In utero constriction of the ductus arteriosus created pulmonary hypertension in fetal lambs (hypertensive fetal lamb [HTFL]). PASMCs isolated 8 days after surgery were assessed for the alteration of protein levels by immunoblots and ROS formation by dihydroethidium and Cell ROX deep red fluorescence. NgBR small interfering RNA and plasmid DNA were used to manipulate NgBR levels. Proliferation and wound healing were assessed by cell counts and scratch recovery assay, respectively. Acute ER stress was induced by tunicamycin. Differences of mitogen-activated protein kinase and Akt pathway activation in HTFL versus control PASMCs were evaluated. Results showed that HTFL PASMCs had decreased NgBR levels and increased proliferation, wound healing, ER stress, and ROS formation compared with controls. Knockdown of NgBR in control PASMCs generated a phenotype similar to HTFL, and overexpression in HTFL restored the defective phenotype to control. Decreased NgBR levels were associated with increased ROS formation in HTFL PASMCs. Subsequently, scavenging ROS decreased proliferation and wound healing. Mechanistically, ROS formation decreases NgBR expression, which induces ER stress. This leads to extracellular signal-regulated kinase pathway activation and PASMC phenotype alteration. Our data suggest that decreased NgBR expression in pulmonary hypertension of the newborn contributes to increased PASMC proliferation and oxidative stress, which lead to the pathogenesis of lung injury.

Nogo-B receptor modulates angiogenesis response of pulmonary artery endothelial cells through eNOS coupling.

Nogo-B, a reticulon-4 isoform, modulates the motility and adhesion of vascular endothelial cells after binding to its receptor, Nogo-B receptor (NgBR). Nogo-B/NgBR pathway contributes to vascular remodeling and angiogenesis, but the role of this pathway in the angiogenesis of developing lungs remains unknown. We previously reported that angiogenesis function of pulmonary artery endothelial cells (PAECs) is impaired by increased reactive oxygen species formation in a fetal lamb model of intrauterine pulmonary hypertension (IPH). Here, we report that Nogo-B/NgBR pathway is altered in IPH, and that decreased NgBR expression contributes to impaired angiogenesis in IPH. We observed a decrease in NgBR levels in lysates of whole lung or PAECs from fetal lambs with IPH compared with controls. Overexpression of NgBR in IPH PAECs rescued the in vitro angiogenesis defects and increased the phosphorylation of both Akt and endothelial nitric oxide synthase at serine(1179) as well as the levels of both manganese superoxide dismutase and GTP cyclohydrolase-1. Consistent with the phenotype of IPH PAECs, knockdown of NgBR in control PAECs decreased the levels of nitric oxide, increased the levels of reactive oxygen species, and impaired in vitro angiogenesis. Our data demonstrate that NgBR mediates PAEC angiogenesis response through the modulation of Akt/endothelial nitric oxide synthase functions, and its decreased expression is mechanistically linked to IPH-related angiogenesis defects in the developing lungs.

Inducible HSP70 regulates superoxide dismutase-2 and mitochondrial oxidative stress in the endothelial cells from developing lungs.

Superoxide dismutase 2 (SOD-2) is synthesized in the cytosol and imported into the mitochondrial matrix, where it is activated and functions as the primary antioxidant for cellular respiration. The specific mechanisms that target SOD-2 to the mitochondria remain unclear. We hypothesize that inducible heat shock protein 70 (iHSP70) targets SOD-2 to the mitochondria via a mechanism facilitated by ATP, and this process is impaired in persistent pulmonary hypertension of the newborn (PPHN). We observed that iHSP70 interacts with SOD-2 and targets SOD-2 to the mitochondria. Interruption of iHSP70-SOD-2 interaction with 2-phenylethylenesulfonamide-µ (PFT-µ, a specific inhibitor of substrate binding to iHSP70 COOH terminus) and siRNA-mediated knockdown of iHSP70 expression disrupted SOD-2 transport to mitochondria. Increasing intracellular ATP levels by stimulation of respiration with CaCl2 facilitated the mitochondrial import of SOD-2, increased SOD-2 activity, and decreased the mitochondrial superoxide (O2(·-)) levels in PPHN pulmonary artery endothelial cells (PAEC) by promoting iHSP70-SOD-2 dissociation at the outer mitochondrial membrane. In contrast, oligomycin, an inhibitor of mitochondrial ATPase, decreased SOD-2 expression and activity and increased O2(·-) levels in the mitochondria of control PAEC. The basal ATP levels and degree of iHSP70-SOD-2 dissociation were lower in PPHN PAEC and lead to increased SOD-2 degradation in cytosol. In normal pulmonary arteries (PA), PFT-µ impaired the relaxation response of PA rings in response to nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine. Pretreatment with Mito-Q, a mitochondrial targeted O2(·-) scavenger, restored the relaxation response in PA rings pretreated with PFT-µ. Our observations suggest that iHSP70 chaperones SOD-2 to the mitochondria. Impaired SOD-2-iHSP70 dissociation decreases SOD-2 import and contributes to mitochondrial oxidative stress in PPHN.

Zinc binding ligands and cellular zinc trafficking: apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1.

Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn(2+) binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial "thermodynamic sink". Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn(2+) chelating agents including N,N,N',N'-(2-pyridylethyl)ethylenediammine (TPEN), EDTA, and [(2,2'-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2-3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK(1) cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn(3)-Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn(3)-Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn(3)-Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK(1) cells to TPEN for 24h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Zn-proteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn(2+).