In this study, a novel lipid vector has been developed for targeted delivery of oligodeoxynucleotides (ODN) to tumors that overexpress folate receptor. This is based on a method developed by Semple et al. (1), which utilizes an ionizable aminolipid (1,2-dioleoyl-3-(dimethylammonio)propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities of polyanionic ODN in lipid vesicles. Folate is incorporated into the lipid vesicles via a distearoylphosphatidylethanolamine−poly(ethylene glycol) (DSPE−PEG) spacer. These vesicles are around 100−200 nm in diameter with an ODN entrapment efficiency of 60−80%. Folate mediated efficient delivery of ODN to KB cells that overexpress folate receptor. Uptake of folate-targeted lipidic ODN by KB cells is about 8−10-fold more efficient than that of lipidic ODN without a ligand or free ODN. This formulation is resistant to serum. Thus, targeted delivery of ODN via this novel lipid vector may have potential in treating tumors that overexpress folate receptors.
Vasoactive properties of sphingosine 1-phosphate (S1P) have been demonstrated by many investigators to vary in systemic vascular beds. These variations appear to reflect differential S1P receptor expression in the vasculature of these tissues. Although S1P has been demonstrated to enhance endothelial barrier function, induce airway hyperresponsiveness, and modulate immune responses in the lung, the pulmonary vasomotor effects of S1P remain poorly defined. In the present study, we sought to define the vasoregulatory effects of S1P in the pulmonary vasculature and to elucidate the underlying mechanisms operative in effecting the response in the intact lung. S1P (10 microM) increased pulmonary vascular resistance (PVR) by 36% in the isolated perfused mouse lung. S1P-induced vasoconstriction was reduced by 64% by concomitant administration of the Rho-kinase inhibitor Y27632 (10 microM). Similarly, the S1P response was attenuated by >50% after S1P(2) receptor antagonism (JTE-013; 10 microM) and in S1P(2) receptor null mice. In contrast, S1P(3) receptor antagonism (VPC23019; 10 microM) had no effect on the contractile response to S1P. Furthermore, we confirmed the role of Rho-kinase as an important regulator of basal vasomotor tone in the isolated perfused mouse lung. These results suggest that S1P is capable of altering pulmonary vascular tone in vivo and may play an important role in the modulation of pulmonary vascular tone both in the normal lung and under pathological conditions.
Unconventional gas drilling (UGD) has enabled extraordinarily rapid growth in the extraction of natural gas. Despite frequently expressed public concern, human health studies have not kept pace. We investigated the association of proximity to UGD in the Marcellus Shale formation and perinatal outcomes in a retrospective cohort study of 15,451 live births in Southwest Pennsylvania from 2007–2010. Mothers were categorized into exposure quartiles based on inverse distance weighted (IDW) well count; least exposed mothers (first quartile) had an IDW well count less than 0.87 wells per mile, while the most exposed (fourth quartile) had 6.00 wells or greater per mile. Multivariate linear (birth weight) or logistical (small for gestational age (SGA) and prematurity) regression analyses, accounting for differences in maternal and child risk factors, were performed. There was no significant association of proximity and density of UGD with prematurity. Comparison of the most to least exposed, however, revealed lower birth weight (3323 ± 558 vs 3344 ± 544 g) and a higher incidence of SGA (6.5 vs 4.8%, respectively; odds ratio: 1.34; 95% confidence interval: 1.10–1.63). While the clinical significance of the differences in birth weight among the exposure groups is unclear, the present findings further emphasize the need for larger studies, in regio-specific fashion, with more precise characterization of exposure over an extended period of time to evaluate the potential public health significance of UGD.
Interleukin (IL)-1beta is an important early mediator of inflammation in pulmonary artery smooth muscle cells. We previously reported that a geranylgeranyltransferase inhibitor elevated basal levels of inducible nitric oxide synthase (iNOS) and enhanced IL-1beta-mediated induction, suggesting that Rac or Rho small G proteins are candidates for antagonism of such induction. In this study, overexpression of constitutively active Rac1 or its dominant negative mutant did not affect IL-1beta induction of iNOS. Alternatively, treatment with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho, was associated with superinduction of iNOS, suggesting an inhibitory role for Rho. IL-1beta activated the three mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38) and the Janus kinase (JAK)-signal transducer and activator of transcription pathways. The former two pathways were not associated with IL-1beta-mediated iNOS induction, whereas the latter two appeared to have inhibitory roles in iNOS expression. These data suggest that a broad intracellular signaling response to IL-1beta in rat pulmonary artery smooth muscle cells results in elevated levels of iNOS that is opposed by the geranylgeranylated small G protein Rho as well as the p38 and JAK2 pathways.
Intracellular safeguarding functions of metallothioneins (MTs) include sequestering transition and heavy metals, scavenging free radicals and protecting against electrophiles. We report that MT protection against Cu-induced cytotoxicity can be reversed and pro-oxidant and pro-apoptotic effects can be induced in HL-60 cells exposed to NO. We demonstrate that in ZnCl2-pretreated HL-60 cells loaded with copper nitrilotriacetate (Cu-NTA), exposure to an NO donor, S-nitroso-N-acetyl penicillamine, resulted in S-nitrosylation and oxidation of MT cysteines. This disruption of MT Cu-binding thiolate clusters caused loosening and release of redox-active Cu, enhanced redox-cycling activity of Cu and increased peroxidation of major classes of membrane phospholipids. We also found that Cu-induced oxidative stress in ZnCl2-pretreated/Cu-NTA-loaded HL-60 cells was accompanied by apoptosis documented by characteristic changes of nuclear morphology, internucleosomal DNA cleavage, externalization of phosphatidylserine, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. We conclude that in Cu-challenged cells, NO can reverse the protective role of MTs and convert them into pro-oxidant, pro-apoptotic implements.
After iron, zinc is the most abundant intracellular metal and Zn i quota is maintained in a narrow range (100‐500 uM) by the activity of zinc importers (SLC39A 1‐14 ) and zinc exporters (SLC30A 1‐10 ). Although virtually all zinc is bound to protein, a chelatable pool of Zn i is capable of participating in intracellular signaling and functions. We used chemical and genetic techniques to reveal a role for decreases in Zn i to affect LPS mediated apoptosis in SPAEC. Early (0.5 to 4.0 hrs) after a proapototic dose (100 ng/ml) of LPS, we noted a decrease in Zn i as revealed by the zinc sensitive fluorophore, FluoZin‐3, via FACS. Confirmation of this relative decrease after LPS was obtained via: a) EPI fluorescence microscopy in live cells that revealed a 60% decrease in Zn i ; and b) 40% decrease of activity of zinc sensitive metal transcription element (MRE) fused to luciferase reporter that was previously transfected into SPAEC. The functional consequences of decreases in Zn i were ascertained by: a) rescuing SPAEC from LPS induced increases in caspase 3/7 by addition of exogenous zinc to normalize potential LPS induced decreases in Zn i ; and b) mimicking effect of LPS on caspase 3/7 activity with zinc chelator, TPEN. We conclude that LPS results in a change in net activity of zinc transporters resulting in decrease in Zn i and this decrease is an important intracellular signaling pathway transducing the apoptotic effects of LPS on SPAEC.
S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a critical link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, respectively. Although low molecular weight S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothlols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and determined, Waconfocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temperature sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low molecular weight S-nitroso-L-cysteine ethyl ester (L-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter molecule. Transnitrosation of FRET-MT by SNO-albumin, but not L-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide Isomerase (csPDI). These results extend the original observations of Ramachandran et al. (Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Proc Natl Acad Sci U S A 98:9539–9544, 2001) and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell molecular targets (e.g. MT).
Significance: Oxygenated polyunsaturated lipids are known to play multi-functional roles as essential signals coordinating metabolism and physiology. Among them are well-studied eicosanoids and docosanoids that are generated via phospholipase A2 hydrolysis of membrane phospholipids and subsequent oxygenation of free polyunsaturated fatty acids (PUFA) by cyclooxygenases and lipoxygenases. Recent Advances: There is an emerging understanding that oxygenated PUFA-phospholipids also represent a rich signaling language with yet-to-be-deciphered details of the execution machinery—oxygenating enzymes, regulators, and receptors. Both free and esterified oxygenated PUFA signals are generated in cells, and their cross-talk and inter-conversion through the de-acylation/re-acylation reactions is not sufficiently explored. Critical Issues: Here, we review recent data related to oxygenated phospholipids as important damage signals that trigger programmed cell death pathways to eliminate irreparably injured cells and preserve the health of multicellular environments. We discuss the mechanisms underlying the trans-membrane redistribution and generation of oxygenated cardiolipins in mitochondria by cytochrome c as pro-apoptotic signals. We also consider the role of oxygenated phosphatidylethanolamines as proximate pro-ferroptotic signals. Future Directions: We highlight the importance of sequential processes of phospholipid oxygenation and signaling in disease contexts as opportunities to use their regulatory mechanisms for the identification of new therapeutic targets.
Abstract Background: The mechanisms by which moderate tidal volume ventilation (MTV) may exacerbate preexisting lung injury remain unclear. We hypothesized that in two hit model (polyinosinic-polycytidylic acid (Poly(I:C)), a synthetic analog of dsRNA and MTV), systemic endotoxemia via gut-lung axis would lead to non-canonical (i.e. caspase-11 dependent) and canonical (caspase-1 dependent) inflammasome activation and programmed necrotic cell death (pyroptosis) contributing to acute lung injury (ALI) in intact mice. Methods: Anesthetized mice were administered Poly(I:C) intratracheally and then 6 h later, they were mechanically ventilated for 4 h with otherwise non-injurious MTV (10ml/kg). Changes in intestinal and alveolar capillary permeability were measured. Further documentation of ALI was assessed by evans blue albumin permeability, protein and IL-1 family concentration in bronchoalveolar lavage fluid (BALF) or plasma, and histopathology in cohorts of wildtype, whole body genetically ablated caspase-11 (caspase-11 -/- ), caspase-1/caspase-11 double knockout (caspase-1/11 -/- ), gasdermin D (GSDMD -/- ), and NLRP3 -/- mice. Results: Non-injurious MTV exacerbated mild Poly(I:C) lung injury including disruption of alveolar-capillary barrier and increased levels of IL-6, HMGB1, IL-1β in BALF and IL-18 in plasma. Combined (Poly(I:C)-MTV) injury was associated with increase in gastrointestinal permeability and endotoxin in plasma and BALF. Poly(I:C)-MTV injury was sensitive to caspase-11 deletion with no further contribution of caspase-1 except for maturation and release of IL-18 (that itself was sensitive to deletion of NLRP3). Combined injury led to large increases in pro-caspase-11 and its cleaved product as well as cleaved product of caspase-1. Genetic ablation of GSDMD attenuated alveolar-capillary disruption and maturation and release of cytokines in combined injury model. Conclusions: The previously noted TLR-4 independent exacerbation of mild Poly(I:C)-induced ALI by otherwise non-injurious MTV is associated with an increase in gut permeability resulting in systemic endotoxemia. The gut-lung axis resulted in activation of pulmonary non-canonical (cytosolic mediated caspase-11 activation) and canonical (caspase-1) inflammasome (NLRP3) mediated ALI in this two hit model resulting in GSDMD sensitive alveolar capillary barrier disruption, pyroptosis (in alveolar macrophages) and cytokine maturation and release (IL-1β; IL-18). Pharmacologic strategies at disrupting communication between gut and lung, inhibition of inflammasomes or effector molecule (GSDMD) in pyroptosis may be useful in ALI.
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