Rux largely restores lungs in Iraq PM-exposed mice, Up-regulating regulatory T-cells (Tregs).

Background Military personnel post-deployment to Iraq and Afghanistan have noted new-onset respiratory illness. This study's primary objective was to further develop an animal model of Iraq Afghanistan War Lung Injury (IAW-LI) and to test a novel class of anti-injury drug called RuX. Methods Particulate Matter (PM) samples were obtained in Iraq then characterized by spectromicroscopy. C57BL/6 mice underwent orotracheal instillation with PM, followed by drinkable treatment with RuX. Lung histology, inspiratory capacity (FlexiVent), thymic/splenic regulatory T cell (Treg) number, and whole-lung genomics were analyzed. Results Tracheal instillation of Iraq PM led to lung septate thickening and lymphocytic inflammation. PM-exposed mice had suppression of thymic/splenic regulatory T-cells (Tregs). Drinking RuX after PM exposure attenuated the histologic lung injury response, improved lung inspiratory capacity, and increased Tregs. Pooled whole lung genomics suggest differences among gene expression of IL-15 among control, PM, and PM + RuX groups. Conclusions RuX, a ruthenium and alpha-lipoic acid complex, attenuates lung injury by improving histology and inspiratory capacity via upregulation of Tregs in Iraq PM-exposed C57BL/6. Plausible genomic effects may involve IL-15 whole lung gene expression.

Denervation-related alterations and biological activity of miRNAs contained in exosomes released by skeletal muscle fibers.

Exosomes are vesicles released by many eukaryotic cells; their cargo includes proteins, mRNA and microRNA (miR) that can be transferred to recipient cells and regulate cellular processes in an autocrine or paracrine manner. While cells of the myoblast lineage secrete exosomes, it is not known whether skeletal muscle fibers (myofibers) release exosomes. In this study, we found that cultured myofibers release nanovesicles that have bilamellar membranes and an average size of 60-130 nm, contain typical exosomal proteins and miRNAs and are taken up by C2C12 cells. miR-133a was found to be the most abundant myomiR in these vesicles while miR-720 was most enriched in exosomes compared to parent myofibers. Treatment of NIH 3T3 cells with myofiber-derived exosomes downregulated the miR-133a targets proteins Smarcd1 and Runx2, confirming that these exosomes have biologically relevant effects on recipient cells. Denervation resulted in a marked increase in miR-206 and reduced expression of miRs 1, 133a, and 133b in myofiber-derived exosomes. These findings demonstrate that skeletal muscle fibers release exosomes which can exert biologically significant effects on recipient cells, and that pathological muscle conditions such as denervation induce alterations in exosomal miR profile which could influence responses to disease states through autocrine or paracrine mechanisms.

Enhanced Mortality to Metastatic Bladder Cancer Cell Line MB49 in Vasoactive Intestinal Peptide Gene Knockout Mice.

To identify if the absence of the vasoactive intestinal peptide (VIP) gene enhances susceptibility to death from metastatic bladder cancer, two strains of mice were injected with MB49 murine bladder cancer cells. The growth and spread of the cancer was measured over a period of 4 weeks in C57BL/6 mice and 5 weeks in VIP knockout (KO) mice. A Kaplan-Meier plot was constructed to compare control C57BL/6 mice and C57BL/6 mice with MB49 vs. VIP KO controls and VIP KO mice with MB49. The wild-type (WT) strain (C57BL/6) contained the VIP gene, while the other strain, VIP knockout backcrossed to C57BL/6 (VIP KO) did not and was thus unable to endogenously produce VIP. VIP KO mice had increased mortality compared to C57BL/6 mice at 4 weeks. The number of ulcers between both groups was not statistically significant. In vitro studies indicated that the presence VIP in high doses reduced MB49 cell growth, as well as macrophage inhibitory factor (MIF), a growth factor in bladder cancer cells. These findings support the concept that VIP may attenuate susceptibility to death from bladder cancer, and that it exerts its effect via downregulation of MIF.

NFATc3 and VIP in Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease.

Idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are both debilitating lung diseases which can lead to hypoxemia and pulmonary hypertension (PH). Nuclear Factor of Activated T-cells (NFAT) is a transcription factor implicated in the etiology of vascular remodeling in hypoxic PH. We have previously shown that mice lacking the ability to generate Vasoactive Intestinal Peptide (VIP) develop spontaneous PH, pulmonary arterial remodeling and lung inflammation. Inhibition of NFAT attenuated PH in these mice suggesting a connection between NFAT and VIP. To test the hypotheses that: 1) VIP inhibits NFAT isoform c3 (NFATc3) activity in pulmonary vascular smooth muscle cells; 2) lung NFATc3 activation is associated with disease severity in IPF and COPD patients, and 3) VIP and NFATc3 expression correlate in lung tissue from IPF and COPD patients. NFAT activity was determined in isolated pulmonary arteries from NFAT-luciferase reporter mice. The % of nuclei with NFAT nuclear accumulation was determined in primary human pulmonary artery smooth muscle cell (PASMC) cultures; in lung airway epithelia and smooth muscle and pulmonary endothelia and smooth muscle from IPF and COPD patients; and in PASMC from mouse lung sections by fluorescence microscopy. Both NFAT and VIP mRNA levels were measured in lungs from IPF and COPD patients. Empirical strategies applied to test hypotheses regarding VIP, NFATc3 expression and activity, and disease type and severity. This study shows a significant negative correlation between NFAT isoform c3 protein expression levels in PASMC, activity of NFATc3 in pulmonary endothelial cells, expression and activity of NFATc3 in bronchial epithelial cells and lung function in IPF patients, supporting the concept that NFATc3 is activated in the early stages of IPF. We further show that there is a significant positive correlation between NFATc3 mRNA expression and VIP RNA expression only in lungs from IPF patients. In addition, we found that VIP inhibits NFAT nuclear translocation in primary human pulmonary artery smooth muscle cells (PASMC). Early activation of NFATc3 in IPF patients may contribute to disease progression and the increase in VIP expression could be a protective compensatory mechanism.

Cystic fibrosis transmembrane conductance regulator dysfunction in VIP knockout mice.

Vasoactive intestinal peptide (VIP), a neuropeptide, controls multiple functions in exocrine tissues, including inflammation, and relaxation of airway and vascular smooth muscles, and regulates CFTR-dependent secretion, which contributes to mucus hydration and local innate defense of the lung. We had previously reported that VIP stimulates the VPAC1 receptor, PKCϵ signaling cascade, and increases CFTR stability and function at the apical membrane of airway epithelial cells by reducing its internalization rate. Moreover, prolonged VIP stimulation corrects the molecular defects associated with F508del, the most common CFTR mutation responsible for the genetic disease cystic fibrosis. In the present study, we have examined the impact of the absence of VIP on CFTR maturation, cellular localization, and function in vivo using VIP knockout mice. We have conducted pathological assessments and detected signs of lung and intestinal disease. Immunodetection methods have shown that the absence of VIP results in CFTR intracellular retention despite normal expression and maturation levels. A subsequent loss of CFTR-dependent chloride current was measured in functional assays with Ussing chamber analysis of the small intestine ex vivo, creating a cystic fibrosis-like condition. Interestingly, intraperitoneal administration of VIP corrected tissue abnormalities, close to the wild-type phenotype, as well as associated defects in the vital CFTR protein. The results show in vivo a primary role for VIP chronic exposure in CFTR membrane stability and function and confirm in vitro data.

Gene Deletion of VIP Leads to Increased Mortality Associated with Progressive Right Ventricular Hypertrophy.

Vasoactive Intestinal Peptide (VIP) knockout mice exhibit asthma, pulmonary hypertension, and left ventricular wall thinning. Humans with these disorders have premature death. We show here that VIP KO mice have reduced survival (100% mortality at 20 months), vs. 100% survival among WT C57BL/6 mice. Moreover, the ratios of weights of right ventricle divided by left ventricle plus septum were progressively increased in VIP KO mice with age. Core temperatures were lower in VIP KO mice when compared to WT littermates, with an associated pro-inflammatory cytokine milieu. Overall, our results indicate that VIP is important for survival in mice. Its absence leads to increased mortality, with progressive right ventricular hypertrophy as a surrogate of pulmonary hypertension, lower body weight, hypothermia, and pro-inflammatory milieu. These studies support VIP as a novel therapeutic agent in pulmonary hypertension.

Iraq dust is respirable, sharp, and metal-laden and induces lung inflammation with fibrosis in mice via IL-2 upregulation and depletion of regulatory T cells.

OBJECTIVES: Determine whether surface dust grab samples taken from a large military base in Iraq are toxic and respirable. METHODS: X-ray diffraction for mineral content, x-ray fluorescence for elemental content, in vivo mouse dust challenges for assessment of histological changes, bronchoalveolar lavage for cytokines, polarizing light microscopy for crystals in lung tissue, and Fluorescence Activated Cell Sorting for cell surface and intracellular markers were utilized. RESULTS: Camp Victory, Iraq dust taken during wartime contains respirable particles 2.5 microns in size, constituting particulate matter air pollution. Dust particles are angular and have sharp edges. Trace metals (including titanium) calcium and silicon are present. Mice with airway instillation of dust have polarizable crystals in lung and septate inflammation. Regulatory T cells (CD4⁺CD25⁺FOXP3⁺) are decreased in thymus and spleen. Interleukin-2 (IL-2) is upregulated in bronchoalveolar lavage. CONCLUSIONS: Respirable Iraq dust leads to lung inflammation in mice similar to that seen in patients with polarizable crystals, which seem to be titanium.

VIP gene deletion in mice causes cardiomyopathy associated with upregulation of heart failure genes.

RATIONALE: Vasoactive Intestinal Peptide (VIP), a pulmonary vasodilator and inhibitor of vascular smooth muscle proliferation, is absent in pulmonary arteries of patients with idiopathic pulmonary arterial hypertension (PAH). We previously determined that targeted deletion of the VIP gene in mice leads to PAH with pulmonary vascular remodeling and right ventricular (RV) dilatation. Whether the left ventricle is also affected by VIP gene deletion is unknown. In the current study, we examined if VIP knockout mice (VIP(-/-)) develop both right (RV) and left ventricular (LV) cardiomyopathy, manifested by LV dilatation and systolic dysfunction, as well as overexpression of genes conducive to heart failure. METHODS: We examined VIP(-/-)and wild type (WT) mice using Magnetic Resonance Imaging (MRI) for evidence of cardiomyopathy associated with biventricular dilation and wall thickness changes. Lung tissue from VIP(-/-) and WT mice was subjected to whole-genome gene microarray analysis. RESULTS: Lungs from VIP(-/-) mice showed overexpression of cardiomyopathy genes: Myh1 was upregulated 224 times over WT, and Mylpf was increased 72 fold. Tnnt3 was increased 105 times and tnnc2 181 fold. Hearts were dilated in VIP(-/-) mice, with thinning of LV wall and increase in RV and LV chamber size, though RV enlargement varied. Weights of VIP(-/-) mice were consistently lower. CONCLUSIONS: Critically-important heart failure-related genes are upregulated in VIP(-/-) mice associated with the spontaneous cardiomyopathy phenotype, involving both left and right ventricles, suggesting that loss of the VIP gene orchestrates a panoply of pathogenic genes which are detrimental to both left and right cardiac homeostasis.

Pharmacogenomics in pulmonary arterial hypertension: Toward a mechanistic, target-based approach to therapy.

Pharmacogenomics is the study of how genetic variations influence the response to drugs, by correlating gene expression with the drug's efficacy and toxicity. This concept has recently been successfully applied in oncology. To test its applicability to PAH, we examined two experimental models of the disease: mice with deletion of the Vasoactive Intestinal Peptide gene (VIP(- /-)); and rats injected with monocrotaline (MCT). Since the two models express comparable phenotypic features, we analyzed their particular gene alterations, with special reference to genes related to pulmonary vasoconstriction, vascular remodeling, and inflammation. We then compared the phenotypic and genotypic responses in each model to treatment with the same drug, VIP. In untreated VIP(-/-) mice there was over-expression of almost all genes promoting vasoconstriction/ proliferation, as well as inflammation, and under-expression of all vasodilator/anti-proliferative genes. As expected, treatment with VIP fully corrected both the key PAH features, and all gene expression alterations. MCT-treated rats showed two distinct sets of alterations. One, similar to that in VIP(- /-) mice, i.e., tended to promote vascular remodeling and inflammation, e.g., up-regulation of myosin polypeptides, procollagen, and some inflammatory genes. The other was a set of opposite alterations that suggested an effort to modulate the PAH, e.g., up-regulation of the VIP and NOS3 genes. In this model, VIP treatment failed to correct many of the genotypic abnormalities, and, in parallel, incompletely corrected the phenotypic changes as well. This preliminary proof-of-concept study demonstrates the importance of genomic information in determining therapeutic outcome, and thus in selecting personalized therapy. Full validation of the merits of pharmacogenomics must await studies of lungs from patients with different forms of PAH.

VIP Regulates the Development & Proliferation of Treg in vivo in spleen.

BACKGROUND: Mounting evidence supports a key role for VIP as an anti-inflammatory agent and promoter of immune tolerance. It suppresses TNF-α and other inflammatory cytokines and chemokines, upregulates anti-inflammatory IL-10, and promotes immune tolerant cells called T regulatory (Treg) cells. VIP KO mice have recently been demonstrated to have spontaneous airway and pulmonary perivascular inflammatory responses, as part of asthma-like and pulmonary hypertension phenotypes, respectively. Both inflammatory responses are correctable with VIP. Focusing on this model, we have now investigated the influence of VIP not only on inflammatory cells but also on Treg cells. METHODS: Using flow cytometric analysis, we examined the relative preponderance of CD25+CD4+ cells and anti-inflammatory Treg cells, in extracts of thymus and spleen from VIP KO mice (5 VIP KO; 5 VIP KO+ VIP; 10 wild-type). This method allowed antibody-based flow cytometric identification of Treg cells using surface markers CD25 and CD4, along with the: 1) intracellular activation marker FoxP3; and 2) Helios, which distinguishes cells of thymic versus splenic derivation. CONCLUSIONS: Deletion of the VIP gene results in: 1) CD25+CD4- cell accumulation in the thymus, which is corrected by VIP treatment; 2) more Treg in thymus lacking Foxp3 expression, suggesting VIP is necessary for immune tolerance; and, 3) a tendency towards deficiency of Treg cells in the spleen, which is normalized by VIP treatment. Treg lacking Helios are induced by VIP intrasplenically rather than by migration from the thymus. These results confirm the dual role of VIP as an anti-inflammatory and immune tolerance-promoting agent.

Vasoactive Intestinal Peptide Knockout (VIP KO) mouse model of sulfite-sensitive asthma: up-regulation of novel lung carbonyl reductase.

BACKGROUND: We earlier reported spontaneous features of asthma in Vasoactive Intestinal Peptide knockout mice (VIP KO): 1) peribronchiolar airway inflammation, with accumulation of lymphocytes and eosinophils, 2) pro-inflammatory cytokine production of IL-5, IL-6, with IFN-γ, and 3) airway hyper-responsiveness to inhaled methacholine. In human asthma, a phenotype with sulfite sensitivity leads to airway inflammation and hyper-responsiveness to inhaled sulfites, and is associated with upregulation of anti-oxidant protein lung carbonyl reductase. For the present experiments, we examined the role of VIP in modulating anti-oxidant genes and their proteins, including lung carbonyl reductase. RESULTS: Four male VIP KO mice and four wild-type age- and gender matched mice had lungs examined for whole genome microarray and a proteomics approach using mass spectrometry. The proteomics analysis revealed that a novel variant of anti-oxidant protein lung carbonyl reductase (car3) was uniquely and markedly elevated in the VIP KO mice. RT-PCR indicated that carbonic anhydrase 3, which is an anti-oxidant protein, was elevated in the VIP KO mice. CONCLUSIONS: These data support the concept that VIP influences the endogenous oxidant/antioxidant balance. One potential implication is that VIP and its analogues may be used to treat inflammatory diseases, including asthma.

VIP and endothelin receptor antagonist: an effective combination against experimental pulmonary arterial hypertension.

BACKGROUND: Pulmonary Arterial Hypertension (PAH) remains a therapeutic challenge, and the search continues for more effective drugs and drug combinations. We recently reported that deletion of the vasoactive intestinal peptide (VIP) gene caused the spontaneous expression of a PH phenotype that was fully corrected by VIP. The objectives of this investigation were to answer the questions: 1) Can VIP protect against PH in other experimental models? and 2) Does combining VIP with an endothelin (ET) receptor antagonist bosentan enhance its efficacy? METHODS: Within 3 weeks of a single injection of monocrotaline (MCT, s.c.) in Sprague Dawley rats, PAH developed, manifested by pulmonary vascular remodeling, lung inflammation, RV hypertrophy, and death within the next 2 weeks. MCT-injected animals were either untreated, treated with bosentan (p.o.) alone, with VIP (i.p.) alone, or with both together. We selected this particular combination upon finding that VIP down-regulates endothelin receptor expression which is further suppressed by bosentan. Therapeutic outcomes were compared as to hemodynamics, pulmonary vascular pathology, and survival. RESULTS: Treatment with VIP, every other day for 3 weeks, begun on the same day as MCT, almost totally prevented PAH pathology, and eliminated mortality for 45 days. Begun 3 weeks after MCT, however, VIP only partially reversed PAH pathology, though more effectively than bosentan. Combined therapy with both drugs fully reversed the pathology, while preventing mortality for at least 45 days. CONCLUSIONS: 1) VIP completely prevented and significantly reversed MCT-induced PAH; 2) VIP was more effective than bosentan, probably because it targets a wider range of pro-remodeling pathways; and 3) combination therapy with VIP plus bosentan was more effective than either drug alone, probably because both drugs synergistically suppressed ET-ET receptor pathway.

17ß-estradiol protects the lung against acute injury: possible mediation by vasoactive intestinal polypeptide.

Beyond their classical role as a class of female sex hormones, estrogens (e.g. 17ß-estradiol) exert important biological actions, both protective and undesirable. We have investigated the ability of estradiol to protect the lung in three models of acute injury induced by 1) oxidant stress due to the herbicide paraquat; 2) excitotoxicity, caused by glutamate agonist N-methyl-d-aspartate; and 3) acute alveolar anoxia. We also assessed the role of estrogen receptors (ER) ERα and ERß and the neuropeptide vasoactive intestinal peptide (VIP) in mediating this protection. Isolated guinea pig or rat lungs were perfused in situ at constant flow and mechanically ventilated. The onset and severity of lung injury were monitored by increases in pulmonary arterial and airway pressures, wet/dry lung weight ratio, and bronchoalveolar lavage fluid protein content. Estradiol was infused into the pulmonary circulation, beginning 10 min before induction of injury and continued for 60-90 min. Lung injury was marked by significant increases in the above measurements, with paraquat producing the most severe, and excitotoxicity the least severe, injury. Estradiol significantly attenuated the injury in each model. Both ER were constitutively expressed and immunohistochemically demonstrable in normal lung, and their selective agonists reduced anoxic injury, the only model in which they were tested. As it protected against injury, estradiol rapidly and significantly stimulated VIP mRNA expression in rat lung. Estradiol attenuated acute lung injury in three experimental models while stimulating VIP gene expression, a known mechanism of lung protection. The up-regulated VIP expression could have partially mediated the protection by estrogen.

Moderate pulmonary arterial hypertension in male mice lacking the vasoactive intestinal peptide gene.

BACKGROUND: Vasoactive intestinal peptide (VIP), a pulmonary vasodilator and inhibitor of vascular smooth muscle proliferation, has been reported absent in pulmonary arteries from patients with idiopathic pulmonary arterial hypertension (PAH). We have tested the hypothesis that targeted deletion of the VIP gene may lead to PAH with pulmonary vascular remodeling. METHODS AND RESULTS: We examined VIP knockout (VIP-/-) mice for evidence of PAH, right ventricular (RV) hypertrophy, and pulmonary vascular remodeling. Relative to wild-type control mice, VIP-/- mice showed moderate RV hypertension, RV hypertrophy confirmed by increased ratio of RV to left ventricle plus septum weight, and enlarged, thickened pulmonary artery and smaller branches with increased muscularization and narrowed lumen. Lung sections also showed perivascular inflammatory cell infiltrates. No systemic hypertension and no arterial hypoxemia existed to explain the PAH. The condition was associated with increased mortality. Both the vascular remodeling and RV remodeling were attenuated after a 4-week treatment with VIP. CONCLUSIONS: Deletion of the VIP gene leads to spontaneous expression of moderately severe PAH in mice during air breathing. Although not an exact model of idiopathic PAH, the VIP-/- mouse should be useful for studying molecular mechanisms of PAH and evaluating potential therapeutic agents. VIP replacement therapy holds promise for the treatment of PAH, and mutations of the VIP gene may be a factor in the pathogenesis of idiopathic PAH.

Mice lacking the VIP gene show airway hyperresponsiveness and airway inflammation, partially reversible by VIP.

The mechanisms leading to asthma, and those guarding against it, are yet to be fully defined. The neuropeptide VIP is a cotransmitter, together with nitric oxide (NO), of airway relaxation, and a modulator of immune and inflammatory responses. NO-storing molecules in the lung were recently shown to modulate airway reactivity and were proposed to have a protective role against the disease. We report here that mice with targeted deletion of the VIP gene spontaneously exhibit airway hyperresponsiveness to the cholinergic agonist methacholine as well as peribronchiolar and perivascular cellular infiltrates and increased levels of inflammatory cytokines in bronchoalveolar lavage fluid. Immunologic sensitization and challenge with ovalbumin generally enhanced the airway hyperresponsiveness and airway inflammation in all mice. Intraperitoneal administration of VIP over a 2-wk period in knockout mice virtually eliminated the airway hyperresponsiveness and reduced the airway inflammation in previously sensitized and challenged mice. The findings suggest that 1) VIP may be an important component of endogenous anti-asthma mechanisms, 2) deficiency of the VIP gene may predispose to asthma pathogenesis, and 3) treatment with VIP or a suitable agonist may offer potentially effective replacement therapy for this disease.