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Abstract Zinc is important. It is the second most abundant trace metal with 2-4 grams in humans. It is an essential trace element, critical for cell growth, development and differentiation, DNA synthesis, RNA transcription, cell division, and cell activation. Zinc deficiency has adverse consequences during embryogenesis and early childhood development, particularly on immune functioning. It is essential in members of all enzyme classes, including over 300 signaling molecules and transcription factors. Free zinc in immune and tumor cells is regulated by 14 distinct zinc importers (ZIP) and transporters (ZNT1-8). Zinc depletion induces cell death via apoptosis (or necrosis if apoptotic pathways are blocked) while sufficient zinc levels allows maintenance of autophagy. Cancer cells have upregulated zinc importers, and frequently increased zinc levels, which allow them to survive. Based on this novel synthesis, approaches which locally regulate zinc levels to promote survival of immune cells and/or induce tumor apoptosis are in order.

Vasoactive properties of sphingosine 1-phosphate (SIP) have been demonstrated by many investigators to vary in systemic vascular beds. These variations appear to reflect differential S IP receptor expression in the vasculature of these tissues. Although S 1P has been demonstrated to enhance endothelial barrier function, induce airway hyperresponsiveness, and modulate immune responses in the lung, the pulmonary vasomotor effects of S 1P remain poorly defined. In the present study, we sought to define the vasoregulatory effects of SIP in the pulmonary vasculature and to elucidate the underlying mechanisms operative in effecting the response in the intact lung. S1P (10 [micro]M) increased pulmonary vascular resistance (PVR) by 36% in the isolated perfused mouse lung. SIP-induced vasoconstriction was reduced by 64% by concomitant administration of the Rho-kinase inhibitor Y27632 (10 [micro]M). Similarly, the S1P response was attenuated by >50% after S1P2 receptor antagonism (JTE-013; 10 [micro]M) and in S1P2 receptor null mice. In contrast, [S1P.sub.3] receptor antagonism (VPC23019; 10 [micro]M) 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. lung; sphingolipid; vasoregulation; pulmonary hypertension doi: 10.1152/ajplung.00233.2009.

Reactive oxygen species have been shown to play a significant role in hyperoxia-induced acute lung injury, in part, by inducing apoptosis of pulmonary endothelium. However, the signaling roles of phospholipid oxidation products in pulmonary endothelial apoptosis have not been studied. Using an oxidative lipidomics approach, we identified individual molecular species of phospholipids involved in the apoptosis-associated peroxidation process in a hyperoxic lung. C57BL/6 mice were killed 72 h after exposure to hyperoxia (100% oxygen). We found that hyperoxia-induced apoptosis (documented by activation of caspase-3 and -7 and histochemical terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of pulmonary endothelium) was accompanied by nonrandom oxidation of pulmonary lipids. Two anionic phospholipids, mitochondria-specific cardiolipin (CL) and extramitochondrial phosphatidylserine (PS), were the two major oxidized phospholipids in hyperoxic lung. Using electrospray ionization mass spectrometry, we identified several oxygenation products in CL and PS. Quantitative assessments revealed a significant decrease of CL and PS molecular species containing [C.sub.18:2], [C.sub.20:4], [C.sub.22:5], and [C.sub.22:6] fatty acids. Similarly, exposure of mouse pulmonary endothelial cells (MLEC) to hyperoxia (95% oxygen; 72 h) resulted in activation of caspase-3 and -7 and significantly decreased the content of CL molecular species containing [C.sub.18:2] and [C.sub.20:4] as well as PS molecular species containing [C.sub.22:5] and [C.sub.22:6]. Oxygenated molecular species were found in the same two anionic phospholipids, CL and PS, in MLEC exposed to hyperoxia. Treatment of MLEC with a mitochondria-targeted radical scavenger, a conjugate of hemi-gramicidin S with nitroxide, XJB-5-131, resulted in significantly lower oxidation of both CL and PS and a decrease in hyperoxia-induced changes in caspase-3 and -7 activation. We speculate that cytochrome c driven oxidation of CL and PS is associated with the signaling role of these oxygenated species participating in the execution of apoptosis and clearance of pulmonary endothelial cells, thus contributing to hyperoxic lung injury. hyperoxia; endothelium; hydroperoxides; apoptosis doi: 10.1152/ajplung.00035.2010.

Matrix metalloproteinases (MMPs) are mediators of lung injury, and their activity has been associated with the development of pulmonary fibrosis. To understand how MMPs regulate the development of pulmonary fibrosis, we examined MMP expression in two strains of mice with differing sensitivities to the fibrosis-inducing drug bleomycin. After a single intratracheal injection of the drug, bleomycin-sensitive C57BL/6 mice showed increased expression for MMPs (-2, -7, -9, -13) at both 7 and 14 days posttreatment compared with the bleomycin-resistant BALB/c strain. In addition, TIMP-1, an endogenous inhibitor of MMPs, was upregulated in the lungs of C57BL/6 mice but not BALB/c mice. We designed two strategies to decrease MMP expression to potentially decrease sensitivity of C57BL/6 mice: 1) we engineered C57BL/6 mice that overexpressed TIMP-1 in their lungs via surfactant protein C (SP-C) promoter; and 2) we inhibited expression of MMPs independent of TIMP-1 by knocking out metallothionein (MT), a critical zinc binding protein. SP-C-TIMP-1 mice reduced MMP expression in response to bleomycin. However, they were equally sensitive to bleomycin as their wild-type counterparts, displaying similar levels of hydroxyproline in the lung tissue. MT null mice displayed decreased lung activity of MMPs with no change in TIMP-1. Nonetheless, there was no difference between the MT null and wild-type control littermates with regards to any of the lung injury parameters measured. We conclude that although TIMP-1 expression is differentially regulated in fibrosis-sensitive and fibrosis-resistant strains, epithelial overexpression of TIMP-1 does not appear to substantially alter fibrotic lung disease in mice. matrix metalloproteinase; pulmonary fibrosis; metallothionein

The recent resurgence of interest in the use of intravital microscopy in lung research is a manifestation of extraordinary progress in visual imaging and optical microscopy. This review evaluates the tools and instrumentation available for a number of imaging modalities, with particular attention to recent technological advances, and addresses recent progress in use of optical imaging techniques in basic pulmonary research. (1) Limitations of existing methods and anticipated future developments are also identified. Although there have also been major advances made in the use of magnetic resonance imaging, positron emission tomography, and X-ray and computed tomography to image intact lungs and while these technologies have been instrumental in advancing the diagnosis and treatment of patients, the purpose of this review is to outline developing optical methods that can be evaluated for use in basic research in pulmonary biology. fluorescent probes; green fluorescent protein; fluorescence resonance energy transfer; electron paramagnetic resonance; electron microscopy

The relationship between cationic lipid-DNA complexes (LPD) and cytokine production levels is examined. Results show that LPD complexes can trigger the production of large quantities of the proinflammatory cytokines that play an important role in complex-induced toxicity. Cytokine production, in this case, is shown to be induced by unmethylated CpG sequences in plasmid DNA. The results suggest that the reduction of CpG-mediated immune response may represent an important approach to the enhancement of cationic lipid-mediated intravenous gene delivery.

Mechanisms by which nitric oxide (NO) is antiapoptotic in pulmonary endothelial cells were investigated using adenoviral, replication-deficient retrovirus (DFG) and S-nitroso-N-acetylpenicillamine. The continuous synthesis of low levels of NO after DFG-inducible NO synthase infection of sheep pulmonary artery epithelial cells inhibited lipopolysaccharide-induced apoptosis. The time-dependent nature of the phenomenon suggested that NO may be inducing expression or activity of other cellular genes.

A study was conducted on the overexpression of metallothionein to tert-butyl hydroperoxide, hyperoxia, or 2,2'-azobis in cultured sheep pulmonary artery endothelial cells. Sphingomyelin, phosphatidylcholine and phosphatidylserine peroxyl oxidation were found to be inhibited by overexpression of metallothionein in endothelial cells after gene transfer. Results demonstrate that metallothionein provides protection against various prooxidant stimuli and lipid peroxidation.

The ability of integrins to prevent DNA strand breakage in pulmonary endothelial cells treated with bleomycin was investigated. Lipopolysaccharide-treated mice were used as experimental models for DNA sedimentation to measure DNA breakage. Results indicated that the degree of integrin activation affected the level of protection provided by integrins against DNA strand breakage. In addition, bleomycin induced 3'-OH DNA breakage in cultured murine lung endothelial cells.

Rats exhibiting lipopolysaccharide (LPS)-induced hypotension were analyzed to determine the effects of heat shock protein induction and nitric oxide synthase (iNOS) inhibition on hypotension. LPS-induced hypotension and vasoplegia were mediated by NO synthetic pathway and iNOS activity. Furthermore, LPS-vasoplegia in rats were inhibited by the expression of HSP 70, heme oxygenase-1 and inhibition of NO synthetic pathway or iNOS activity.

Cultured endothelial cells of sheep pulmonary artery were analyzed to determine the effects of nitric oxide (NO) on intracellular heme and iron metabolism. NO decreases the concentration of heme-iron content in sheep epithelial cells by inducing the expression of the heme oxygenase-1 (HO-1) gene. However, HO-1 gene expression increased the levels of iron-responsive genes such as mitochondrial aconitase and ferritin.

The enzyme nitric oxide synthase (NOS) catalyzes the synthesis of pulmonary NO which provide protection against injury and disease. In a study of cultured rat pulmonary artery smooth muscle cells (RPASMC), treatment of these cells with interleukin-1beta (IL-1beta) stimulated transcription of intracellular NOS. IL-1beta also caused increases in the activity of the 5'-flanking promoter region of the NOS gene. These increases were sustained when the promoter fragment was not shorter than -242 bp. These results suggest that IL-1beta induces NOS gene expression in RPASMC.

The destructive effects of bacterial lipopolysaccharides on the integrity of the DNA strand is counteracted by the basement membrane protein integrin. DNA strand breakage by lipopolysaccharides normally initiate cell death. When integrins are activated in the presence of these toxins, they cause a rearrangement in the cytoskeleton and induce the activity of enzymes and proteases that would inhibit cell death and allow the DNA to repair itself.

Rat pulmonary artery smooth muscle cells in culture were exposed to sodium arsenite or heat to examine the effect of heat shock proteins on the expression of inducible nitric oxide synthase (iNOS). The results show that heat shock proteins suppressed interleukin-1-beta-mediated iNOS gene expression. This may be a mechanism to prevent pr reduce inflammatory lung damage caused by overproduction of nitric oxide.

Study on the produced lipopolysaccharide (LPS) apoptosis in sheep pulmonary artery endothelial cells (SPAEC) showed that SPAEC cultured on collagen was protected from LPS-induced apoptosis and suppressed the nuclear damage. The inactivation of LPS is seen in the role of this protection. This indicated that LPS-induced apoptosis occurs after the genotoxic damage which is suppressed by the extracellular matrix. The influence of the extracellular matrix on LPS-induced apoptosis in SPAEC is evaluated.

The combination of cytokines, zymosan activated serum (ZAS) and LPS stimulates the production of high concentrations of NO oxidation products in lung and rat epithelial cells. The inflammatory mediator induced NO production by alveolar epithelium also reveals the new mechansims of the pulmonary host defense systems.

Nitric oxide production induced by cytokine and lipopolysaccharide (LPS) production requires the synthesis of tetrahydrobiopterin (BH4) in rat pulmonary artery smooth muscle cells (RPASM). Tetrahydrobiopterin synthesis regulates inducible nitric oxide synthase (iNOS) in RPASM. 2,4-diamino-6-hydroxypyrimidine (DAHP) prevents the accumulation of LPS and cytokine-induced intracellular biopterins. Sepiapterin reverses DAHP's influence on LPS and biopterins and also produces BH4 through a GTP-cyclohydrolase-independent salvage pathway.

Cultured rat vascular smooth muscle cells from peripheral and proximal lung express mRNA in the presence of serotonin type 2 (5-HT2) receptor. A ketanserin-sensitive 5-HT-induced enhancement in DNA synthesis accompanies the functional expression of this gene. The mitosis-inducing effect of 5-HT is reduced by fluoxetine, a 5-HT transport blocker, indicating the involvement of 5-HT transport in this event. The receptor-mediated processes of 5-HT are also influenced by 5-HT.