Bruce R. Pitt

Cytochrome c/cardiolipin relations in mitochondria: a kiss of death

Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. During apoptosis, a mitochondria-specific phospholipid, cardiolipin (CL), interacts with cytochrome c (cyt c) to form a peroxidase complex that catalyzes CL oxidation; this process plays a pivotal role in the mitochondrial stage of the execution of the cell death program. This review is focused on redox mechanisms and essential structural features of cyt cs conversion into a CL-specific peroxidase that represent an interesting and maybe still unique example of a functionally significant ligand change in hemoproteins. Furthermore, specific characteristics of CL in mitochondria--its asymmetric transmembrane distribution and mechanisms of collapse, the regulation of its synthesis, remodeling, and fatty acid composition--are given significant consideration. Finally, new concepts in drug discovery based on the design of mitochondria-targeted inhibitors of cyt c/CL peroxidase and CL peroxidation with antiapoptotic effects are presented.

The Inhibitory Role of CpG Immunostimulatory Motifs in Cationic Lipid Vector-Mediated Transgene Expression in Vivo

We have previously reported that intravenous administration of cationic lipid-protamine-DNA complexes (LPD) induces production of large quantities of proinflammatory cytokines that are toxic and cause inhibition of transgene expression. Cytokine induction appears to be mediated by the unmethylated CpG sequences since methylation of plasmid DNA significantly decreases the cytokine levels. In this study, the inhibitory role of CpG in lipid-mediated gene transfer was further investigated using chemically well-defined, CpG-containing oligodeoxynucleotides (ODNs). Injection (intravenous) of ODNs formulated in LPD into mice triggered production of proinflammatory cytokines including interferon gamma and TNF-alpha. The potency of CpG-containing ODNs in cytokine induction was affected by its flanking sequences and was significantly reduced when CpG was methylated. Preinjection of ODN-containing LPD led to inhibition of transgene expression in lungs after a subsequent injection of LPD containing plasmid expression vector with luciferase gene. The degree of inhibition correlated with the levels of ODN-triggered cytokines. Finally, intraperitoneal injection of dexamethasone suppressed LPD-induced cytokine production, and led to significantly higher levels of transgene expression on both first and second injection. These studies suggest that mutation of potent CpG motifs in plasmid DNA together with the use of immune suppression agent may represent an effective approach to improve cationic lipid-mediated gene transfer to the lung.

Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors

Cationic lipid-mediated intravenous gene delivery shows promise in treating pulmonary diseases including lung tumor metastases, pulmonary hypertension, and acute respiratory distress syndrome. Nevertheless, clinical applications of cationic lipidic vectors via intravenous administration are limited by their transient gene expression. In addition, repeated dosing is not effective at frequent intervals. In an effort to elucidate the mechanism of gene inactivation, we report in this study that cationic lipid-protamine-DNA (LPD) complexes, but not each component alone, can induce a high level of cytokine production, including interferon-γ and tumor necrosis factor-α. Furthermore, we demonstrate that LPD administration triggers apoptosis in the lung, a phenomenon that may be mediated in part by the two cytokines. Treatment of mice with antibodies against the two cytokines prolongs the duration of gene expression and also improves lung transfection on a second administration of LPD. Although the mechanism underlying LPD-induced cytokine production is unclear, methylation of the DNA significantly decreased the level of both interferon-γ and tumor necrosis factor-α, suggesting that unmethylated CpG sequences in plasmid DNA play an important role. These data suggest that decreasing the CpG-mediated immune response while not affecting gene expression may be a useful therapeutic strategy to improve cationic lipid-mediated intravenous gene delivery to the lung.

Adenoviral transfer of the inducible nitric oxide synthase gene blocks endothelial cell apoptosis

BACKGROUND We have previously reported that vascular inducible nitric oxide synthase (iNOS) gene transfer inhibits injury-induced intimal hyperplasia in vitro and in vivo. One mechanism by which NO may prevent intimal hyperplasia is by preserving the endothelium or promoting its regeneration. To study this possibility we examined the effect of iNOS gene transfer on endothelial cell (EC) proliferation and viability. METHODS An adenoviral vector (AdiNOS) containing the human iNOS cDNA was constructed and used to infect cultured sheep arterial ECs. NO production was measured, and the effects of continuous NO exposure on EC proliferation, viability, and apoptosis were evaluated. RESULTS AdiNOS-infected ECs produced 25- to 100-fold more NO than control (AdlacZ) infected cells as measured by nitrite accumulation. This increased NO synthesis did not inhibit EC proliferation as reflected by tritiated thymidine incorporation. Chromium 51 release assay revealed that EC viability was also unaffected by AdiNOS infection and NO synthesis. In addition, prolonged exposure to NO synthesis did not induce EC apoptosis. Instead, NO inhibited lipopolysaccharide-induced apoptosis in these cells by reducing caspase-3-like protease activity. CONCLUSIONS Vascular iNOS gene transfer, while inhibiting smooth muscle cell proliferation, does not impair EC mitogenesis or viability. Augmented NO synthesis may also protect ECs against apogenic stimuli such as lipopolysaccharide. Therefore iNOS gene transfer may promote endothelial regeneration and can perhaps accelerate vascular healing.

Downregulation of Endothelin-1 by Farnesoid X Receptor in Vascular Endothelial Cells

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidney, adrenals, and intestine. FXR may play an important role in the pathogenesis of cardiovascular diseases via regulating the metabolism and transport of cholesterol. In this study, we report that FXR is also expressed in rat pulmonary artery endothelial cells (EC), a “nonclassical” bile acid target tissue. FXR is functional in EC, as demonstrated by induction of its target genes such as small heterodimer partner (SHP) after treatment with chenodeoxycholic acid, a FXR agonist. Interestingly, activation of FXR in EC led to downregulation of endothelin (ET)-1 expression. Reporter assays showed that activation of FXR inhibited transcriptional activation of the human ET-1 gene promoter and also repressed the activity of a heterologous promoter driven by activator protein (AP)-1 response elements. Electrophoretic mobility-shift and chromatin immunoprecipitation assays indicated that FXR reduced the binding activity of AP-1 transcriptional factors, suggesting that FXR may suppress ET-1 expression via negatively interfering with AP-1 signaling. These studies suggest that FXR may play a role in endothelial homeostasis and may serve as a novel molecular target for manipulating ET-1 expression in vascular EC.

The protein thiol metallothionein as an antioxidant and protectant against antineoplastic drugs

Metallothioneins (MTs) are major zinc-binding protein thiols that are readily inducible and whose functions remain unclear. Recent evidence supports a role for MT as an antioxidant. Mechanisms underlying this function may include direct interception of free radicals, complexation of redox sensitive transition metals, altered zinc homeostasis or interaction with glutathione (GSH). MT overexpression after direct gene transfer in cultured cells, decreases cytotoxicity, to partially reduce reactive oxygen and nitrogen species and markedly attenuates intracellular oxidation of reporter molecules including dichlorofluorescein and cis-parinaric acid. Conversely, enhanced intracellular oxidation is seen in cells derived from mice lacking both functional MTI and MTII genes. GSH levels are unaffected in MT null cells relative to wildtype, suggesting the antioxidant function of MT is independent of GSH. In tumor cells there is at least a 400-fold range in MT levels and a 10-fold difference in the ratio of nuclear to cytoplasmic distribution. No correlation exists between MT levels and GSH levels demonstrating the autonomous regulation of intracellular thiol pools. This may be important for cancer chemotherapies since MT overexpression is seen in human tumor cells with acquired drug resistance. The authors found no evidence for altered MT isoform profiles in drug resistant cells that overexpress MT. Recent evidence suggests MT subcellular location may dictate functionality and MT may help determine the threshold for apoptosis. Thus, MT is a stress-inducible protein with antioxidant attributes that may participate independently or in conjunction with GSH to protect cells against injurious agents.

Polycations increase the efficiency of adenovirus-mediated gene transfer to epithelial and endothelial cells in vitro

Recombinant adenoviruses are being developed for gene therapy for cystic fibrosis and other lung diseases, and for prevention and treatment of vascular thrombosis. A major limitation to the clinical utility of adenoviruses is the low efficiency of gene transfer achieved in vivo. In addition, little is known about the initial interactions between adenoviruses and the target cell. To address the hypothesis that the negative charge presented by membrane glycoproteins reduces the efficiency of adenovirus-mediated gene transfer, primary cultures of human airway, Madin–Darby canine kidney cells, an immortalized cystic fibrosis airway epithelial cell line, and primary cultures of sheep pulmonary artery endothelium were infected with recombinant adeno- virus containing the E. coli lacZ reporter gene (Ad2βgal2) in the presence of various polyions. For each cell type, adsorption of Ad2βgal2 in the presence of the polycations polybrene, protamine, DEAE-dextran, and poly-L-lysine significantly increased the percentage of cells that express lacZ. The polyanion heparin did not significantly alter gene transfer efficiency, but completely abrogated the effects of polycations. These data provide evidence that negatively charged moieties on the cell surface reduce the efficiency of adenovirus-mediated gene transfer, and that alteration of the charge interaction between adenoviruses and the cell surface may improve the potential clinical application of these vectors.

Mitochondrial targeting of electron scavenging antioxidants: Regulation of selective oxidation vs random chain reactions

Effective regulation of highly compartmentalized production of reactive oxygen species and peroxidation reactions in mitochondria requires targeting of small molecule antioxidants and antioxidant enzymes into the organelles. This review describes recently developed approaches to mitochondrial targeting of small biologically active molecules based on: (i) preferential accumulation in mitochondria because of their hydrophobicity and positive charge (hydrophobic cations), (ii) binding with high affinity to an intra-mitochondrial constituent, and (iii) metabolic conversions by specific mitochondrial enzymes to reveal an active entity. In addition, targeted delivery of antioxidant enzymes via expression of leader sequences directing the proteins into mitochondria is considered. Examples of successful antioxidant and anti-apoptotic protection based on the ability of targeted cargoes to inhibit cytochrome c-catalyzed peroxidation of a mitochondria-specific phospholipid cardiolipin, in vitro and in vivo are presented. Particular emphasis is placed on the employment of triphenylphosphonium- and hemi-gramicidin S-moieties as two effective vehicles for mitochondrial delivery of antioxidants.

Randomized indomethacin trial for prevention of intraventricular hemorrhage in very low birth weight infants.

We admitted 48 preterm neonates (600 to 1250 gm birth weight, normal 6-hour echoencephalograms) to a randomized prospective indomethacin or placebo trial for the prevention of neonatal intraventricular hemorrhage. Beginning at 6 postnatal hours, indomethacin or placebo was administered intravenously every 12 hours for a total of five doses. Cardiac ultrasound studies to assess the status of the ductus arteriosus were performed at 6 postnatal hours and on day 5. Urinary output, serum electrolytes, and renal and clotting functions were monitored. No differences in birth weight, gestational age, Apgar scores, or ventilatory needs were noted between the two groups. Six infants given indomethacin had intraventricular hemorrhage, compared to 14 control infants (P = 0.02). The indomethacin-treated group had significant decreases in serum prostaglandin values 30 hours after the initiation of therapy. The overall incidence of patent ductus arteriosus was 82% at 6 postnatal hours; 84% of the indomethacin-treated infants experienced closure of the ductus, compared to 60% of the placebo-treated patients. Closure of the ductus was not related to incidence of intraventricular hemorrhage. We speculate that indomethacin may provide some protection against neonatal intraventricular hemorrhage by acting on the cerebral microvasculature.

Inhibiting geranylgeranylation blocks growth and promotes apoptosis in pulmonary vascular smooth muscle cells

The activity of small GTP-binding proteins is regulated by a critical step in posttranslational processing, namely, the addition of isoprenoid lipids farnesyl and geranylgeranyl, mediated by the enzymes farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I), respectively. We have developed compounds that inhibit these enzymes specifically and in this study sought to determine their effects on smooth muscle cells (SMC) from the pulmonary microvasculature. We found that the GGTase I inhibitor GGTI-298 suppressed protein geranylgeranylation and blocked serum-dependent growth as measured by thymidine uptake and cell counts. In the absence of serum, however, GGTI-298 induced apoptosis in these cells as measured by both DNA staining and flow cytometry. The FTase inhibitor FTI-277 selectively inhibited protein farnesylation but had a minor effect on growth and no effect on apoptosis. To further investigate the role of geranylgeranylated proteins in apoptosis, we added the cholesterol synthesis inhibitor lovastatin, which inhibits the biosynthesis of farnesyl and geranylgeranyl pyrophosphates. This also induced apoptosis, but when geranylgeraniol was added to replenish cellular pools of geranylgeranyl pyrophosphate, apoptosis was reduced to baseline. In contrast, farnesol achieved only partial rescue of the cells. These results imply that geranylgeranylated proteins are required for growth and protect SMC against apoptosis. GGTase I inhibitors may be useful in preventing hyperplastic remodeling and may have the potential to induce the apoptotic regression of established vascular lesions.