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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.

There is a reduction in the sensitivity of NIH 3T3 cells to the nuclear DNA-damaging effects of nitric oxide (NO) due to the overexpression of metallothionein (MT). Electronic paramagnetic resonance spectroscopy shows that MT may be a thiol donor in forming iron-dinitrosyl complexes. Cells transfected with mouse MT-I gene (NIH 3T3/MT) showed a four-fold increase in MT compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells are more resistant than the NIH 3T3/TM cells to cytotoxic effects of the NO donor S-nitrosoacetylpenicillamine.

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.

Interleukin (IL)-1 [Beta] 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-l [Beta]-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-1 [Beta] 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-1 [Beta] 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-1 [Beta]-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-1 [Beta] 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. inducible nitric oxide synthase; interleukin-l [Beta]; Rho; mitogen-activated protein kinase

Role of zinc in pulmonary endothelial cell response to oxidative stress. Am J Physiol Lung Cell Mol Physiol 281: L243-L249, 2001.--Although zinc is a well-known inhibitor of apoptosis, it may contribute to oxidative stress-induced necrosis. We noted that N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN; [is greater than] 10 [micro]M), a zinc chelator, quenched fluorescence of the zinc-specific fluorophore Zinquin and resulted in an increase in spontaneous apoptosis in cultured sheep pulmonary artery endothelial cells (SPAECs). Addition of exogenous zinc (in the presence of pyrithione, a zinc ionophore) to the medium of SPAECs caused an increase in Zinquin fluorescence and was associated with a concentration-dependent increase in necrotic cell death. Exposure of SPAECs to TPEN (10 [micro]M) resulted in enhanced apoptosis after lipopolysaccharide or complete inhibition oft-butyl hydroperoxide (tBH)-induced necrosis. We further investigated the role of two zinc-dependent enzymes, poly(ADP-ribose) polymerase (PARP) and protein kinase (PK) C, in tBH toxicity, tBH toxicity was only affected by the PARP inhibitors 4-amino-1, 8-naphthalimide or 3-aminobenzamide over a narrow range, whereas the PKC inhibitors bisindolylmaleimide and staurosporine significantly reduced tBH toxicity, tBH caused translocation of PKC to the plasma membrane of SPAECs that was partially inhibited by TPEN. Thus pulmonary endothelial cell zinc inhibits spontaneous and lipopolysaccharide-dependent apoptosis but contributes to tBH-induced necrosis, in part, via a PKC-dependent pathway. endothelium; apoptosis; necrosis; protein kinase C

Lung redox homeostasis: emerging concepts. Am J Physiol Lung Cell Mol Physiol 279: L413-L417, 2000.--This symposium was organized to present some aspects of current research pertaining to lung redox function. Focuses of the symposium were on roles of pulmonary endothelial NADPH oxidase, xanthine oxidase (XO)/xanthine dehydrogenase (XDH), heme oxygenase (HO), transplasma membrane electron transport (TPMET), and the zinc binding protein metallothionein (MT) in the propagation and/or protection of the lung or other organs from oxidative injury. The presentations were chosen to reflect the roles of both intracellular (metallothionein, XO/XDH, and HO) and plasma membrane (NADPH oxidase, XO/XDH, and unidentified TPMET) redox proteins in these processes. Although the lung endothelium was the predominant cell type under consideration, at least some of the proposed mechanisms operate in or affect other cell types and organs as well. endothelium; oxidation-reduction; oxidative stress

Recent in vitro studies suggest that the oxidoreductive capacity of metal thiolate clusters in metallothionein (MT) contributes to intracellular zinc homeostasis. We used fluorescence-based techniques to address this hypothesis in intact endothelial cells, focusing on the contributory role of the important redox signaling molecule, nitric oxide. Microspectrofluorometry with Zinquin revealed that the exposure of cultured sheep pulmonary artery endothelial cells to S-nitrosocysteine resulted in the release of N,N,N',N'-tetrakis(2 [multiplied by] pyridylmethyl) ethylendiamine (TPEN) chelatable zinc. Cultured sheep pulmonary artery endothelial cells were transfected with a plasmid expression vector suitable for fluorescence resonance energy transfer containing the cDNA of MT sandwiched between two mutant green fluorescent proteins. The exposure of cultured sheep pulmonary artery endothelial cells transfected with this chimera to nitric oxide donors or to agents that increased cytoplasmic [Ca.sup.2+] via endogenously generated nitric oxide decreased the efficiency of fluorescence resonance energy transfer in a manner consistent with the release of metal (Zn) from MT. A physiological role for this interaction in intact tissue was supported by the lack of myogenic reflex in resistance arteries of MT knockout mice unless endogenous nitric oxide synthesis was blocked. These data suggest an important role for metal thiolate clusters of MT in nitric oxide signaling in the vascular wall. J. Nutr. 130: 1467S--1470S, 2000. KEY WORDS: * metallothionein * zinc * nitric oxide * endothelium * myogenic reflex

Angiogenesis and morphogenesis of murine fetal distal lung in an allograft model. Am J Physiol Lung Cell Mol Physiol 278: L1000-L1007, 2000.--Neovascularization is crucial to lung morphogenesis; however, factors determining vessel growth and formation are poorly understood. The goal of our study was to develop an allograft model that would include maturation of the distal lung, thereby ultimately allowing us to study alveolar development, including microvascular formation. We transplanted 14-day gestational age embryonic mouse lung primordia subcutaneously into the back of nude mice for 3.5-14 days. Lung morphogenesis and neovascularization were evaluated by light microscopy, in situ hybridization, and immunohistochemical techniques. Embryonic 14-day gestational age control lungs had immature structural features consistent with pseudoglandular stage of lung development. In contrast, 14 days after subcutaneous transplantation of a 14-day gestational age lung, the allograft underwent significant structural morphogenesis and neovascularization. This was demonstrated by continued neovascularization and cellular differentiation, resulting in mature alveoli similar to those noted in the 2-day postnatal neonatal lung. Confirmation of maturation of the allograft was provided by progressive type II epithelial cell differentiation as evidenced by enhanced local expression of mRNA for surfactant protein C and a threefold (P [is less than] 0.008) increase in vessel formation as determined by immunocytochemical detection of platelet endothelial cell adhesion molecule-1 expression. Using the tyrosine kinase Flk-1 receptor (flk-1) LacZ transgene embryos, we determined that the neovascularization within the allograft was from the committed embryonic lung endothelium. Therefore, we have developed a defined murine allograft model that can be used to study distal lung development, including neovascularization. The model may be useful when used in conjunction with an altered genetic background (knockout or knock in) of the allograft and has the further decided advantage of bypassing placental barriers for introduction of pharmacological agents or DNA directly into the lung itself. pulmonary microvasculature; lung development; embryo; vasculogenesis; surfactant protein C; platelet endothelial cell adhesion molecule-1

Li, Song, Yadi Tan, Ekapop Viroonchatapan, Bruce R. Pitt, and Leaf Huang. Targeted gene delivery to pulmonary endothelium by anti-PECAM antibody. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L504-L511, 2000.--To achieve efficient systemic gene delivery to the lung with minimal toxicity, a vector was developed by chemically conjugating a cationic polymer, polyethylenimine (PEI), with anti-platelet endothelial cell adhesion molecule (PECAM) antibody (Ab). Transfection of mouse lung endothelial cells with a plasmid expression vector with cDNA to luciferase (pCMVL) complexed with anti-PECAM Ab-PEI conjugate was more efficient than that with PEI-pCMVL complexes. Furthermore, the anti-PECAM Ab-PEI conjugate mediated efficient transfection at lower charge plus-to-minus ratios. Conjugation of PEI with a control IgG (hamster IgG) did not enhance transfection of mouse lung endothelial cells, suggesting that the cellular uptake of anti-PECAM Ab-PEI-DNA complexes and subsequent gene expression were governed by a receptor-mediated process rather than by a nonspecific charge interaction. Conjugation of PEI with anti-PECAM Ab also led to significant improvement in lung gene transfer to intact mice after intravenous administration. The increase in lung transfection was associated with a decrease compared with PEI-pCMVL with respect to circulating proinflammatory cytokine (tumor necrosis factor-[Alpha]) levels. These results indicate that targeted gene delivery to the lung endothelium is an effective strategy to enhance gene delivery to the pulmonary circulation while simultaneously reducing toxicity. cationic polymer; plasmid deoxyribonucleic acid; gene transfer; targeting; toxicity

Johnson, Bruce A., Bruce R. Pitt, and Paul Davies. Pulmonary arterial smooth muscle cells modulate cytokine- and LPS-induced cytotoxicity in endothelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L460-L468, 2000. --Cytokines and lipopolysaccharide (LPS) are known to be injurious to vascular endothelial cells (ECs), but the influence of adjacent vascular smooth muscle cells (SMCs) on this injury is unknown. Exposure of cultured rat (RPMECs) or human (HPMECs) pulmonary microvascular ECs on tissue culture plastic to a mixture of cytokines (interleukin-1[Beta], tumor necrosis factor-[Alpha], and interferon-[Gamma]) and LPS (cytomix) resulted in a significant increase in [sup.51]Cr release to 35-40%. When unstimulated RPMECs were cocultured with cytomix-pretreated rat pulmonary microvascular SMCs (RPMSMCs) there was an increase in [sup.51]Cr release to 8.4%, which was nitric oxide dependent. However, when RPMECs or HPMECs were stimulated in direct contact with their respective SMCs. rather than a further increase in cytomix-induced injury (e.g., [is greater than] 35-40%), [sup.51]Cr release decreased to [is less than] 10%. This cytoprotection was fully reproduced with fixed RPMSMCs, and partially reproduced by plating HPMECs on gelatin. These data show that the direct toxicity of a cytokine and endotoxin mixture on cultured ECs can be reduced by contact with vascular smooth muscle. pulmonary microcirculation; pulmonary endothelium; pulmonary vascular smooth muscle; nitric oxide; human; rat; cytokines; endotoxin; lipopolysaccharide

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic [Ca.sup.2+] act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice ([MT.sup.-/-]) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.