Ventilation defects observed with hyperpolarized 3He magnetic resonance imaging in a mouse model of acute lung injury.

Regions of diminished ventilation are often evident during functional pulmonary imaging studies, including hyperpolarized gas magnetic resonance imaging (MRI), positron emission tomography, and computed tomography (CT). The objective of this study was to characterize the hypointense regions observed via (3)He MRI in a murine model of acute lung injury. LPS at doses ranging from 15-50 µg was intratracheally administered to C57BL/6 mice under anesthesia. Four hours after exposure to either LPS or saline vehicle, mice were imaged via hyperpolarized (3)He MRI. All images were evaluated to identify regions of hypointense signals. Lungs were then characterized by conventional histology, or used to obtain tissue samples from regions of normal and hypointense (3)He signals and analyzed for cytokine content. The characterization of (3)He MRI images identified three distinct types of hypointense patterns: persistent defects, atelectatic defects, and dorsal lucencies. Persistent defects were associated with the administration of LPS. The number of persistent defects depended on the dose of LPS, with a significant increase in mean number of defects in 30-50-µg LPS-dosed mice versus saline-treated control mice. Atelectatic defects predominated in LPS-dosed mice under conditions of low-volume ventilation, and could be reversed with deep inspiration. Dorsal lucencies were present in nearly all mice studied, regardless of the experimental conditions, including control animals that did not receive LPS. A comparison of (3)He MRI with histopathology did not identify tissue abnormalities in regions of low (3)He signal, with the exception of a single region of atelectasis in one mouse. Furthermore, no statistically significant differences were evident in concentrations of IL-1ß, IL-6, macrophage inflammatory protein (MIP)-1α, MIP-2, chemokine (C-X-C motif) ligand 1 (KC), TNFα, and monocyte chemotactic protein (MCP)-1 between hypointense and normally ventilated lung regions in LPS-dosed mice. Thus, this study defines the anatomic, functional, and biochemical characteristics of ventilation defects associated with the administration of LPS in a murine model of acute lung injury.

Prostaglandin E2 protects murine lungs from bleomycin-induced pulmonary fibrosis and lung dysfunction.

Prostaglandin E(2) (PGE(2)) is a lipid mediator that is produced via the metabolism of arachidonic acid by cyclooxygenase enzymes. In the lung, PGE(2) acts as an anti-inflammatory factor and plays an important role in tissue repair processes. Although several studies have examined the role of PGE(2) in the pathogenesis of pulmonary fibrosis in rodents, results have generally been conflicting, and few studies have examined the therapeutic effects of PGE(2) on the accompanying lung dysfunction. In this study, an established model of pulmonary fibrosis was used in which 10-12-wk-old male C57BL/6 mice were administered a single dose (1.0 mg/kg) of bleomycin via oropharyngeal aspiration. To test the role of prostaglandins in this model, mice were dosed, via surgically implanted minipumps, with either vehicle, PGE(2) (1.32 µg/h), or the prostacyclin analog iloprost (0.33 µg/h) beginning 7 days before or 14 days after bleomycin administration. Endpoints assessed at 7 days after bleomycin administration included proinflammatory cytokine levels and measurement of cellular infiltration into the lung. Endpoints assessed at 21 days after bleomycin administration included lung function assessment via invasive (FlexiVent) analysis, cellular infiltration, lung collagen content, and semiquantitative histological analysis of the degree of lung fibrosis (Ashcroft method). Seven days after bleomycin administration, lymphocyte numbers and chemokine C-C motif ligand 2 expression were significantly lower in PGE(2)- and iloprost-treated animals compared with vehicle-treated controls (P < 0.05). When administered 7 days before bleomycin challenge, PGE(2) also protected against the decline in lung static compliance, lung fibrosis, and collagen production that is associated with 3 wk of bleomycin exposure. However, PGE(2) had no therapeutic effect on these parameters when administered 14 days after bleomycin challenge. In summary, PGE(2) prevented the decline in lung static compliance and protected against lung fibrosis when it was administered before bleomycin challenge but had no therapeutic effect when administered after bleomycin challenge.

Modulation of allergic airway inflammation by the oral pathogen Porphyromonas gingivalis.

Accumulating evidence suggests that bacteria associated with periodontal disease may exert systemic immunomodulatory effects. Although the improvement in oral hygiene practices in recent decades correlates with the increased incidence of asthma in developed nations, it is not known whether diseases of the respiratory system might be influenced by the presence of oral pathogens. The present study sought to determine whether subcutaneous infection with the anaerobic oral pathogen Porphyromonas gingivalis exerts a regulatory effect on allergic airway inflammation. BALB/c mice sensitized and subsequently challenged with ovalbumin exhibited airway hyperresponsiveness to methacholine aerosol and increased airway inflammatory cell influx and Th2 cytokine (interleukin-4 [IL-4], IL-5, and IL-13) content relative to those in nonallergic controls. Airway inflammatory cell and cytokine contents were significantly reduced by establishment of a subcutaneous infection with P. gingivalis prior to allergen sensitization, whereas serum levels of ovalbumin-specific IgE and airway responsiveness were not altered. Conversely, subcutaneous infection initiated after allergen sensitization did not alter inflammatory end points but did reduce airway responsiveness in spite of increased serum IgE levels. These data provide the first direct evidence of a regulatory effect of an oral pathogen on allergic airway inflammation and responsiveness. Furthermore, a temporal importance of the establishment of infection relative to allergen sensitization is demonstrated for allergic outcomes.

Overexpression of cytochrome P450 epoxygenases prevents development of hypertension in spontaneously hypertensive rats by enhancing atrial natriuretic peptide.

Cytochrome P450 (P450)-derived epoxyeicosatrienoic acids (EETs) exert well recognized vasodilatory, diuretic, and tubular fluid-electrolyte transport actions that are predictive of a hypotensive effect. The study sought to determine the improvement of hypertension and cardiac function by overexpressing P450 epoxygenases in vivo. Long-term expression of CYP102 F87V or CYP2J2 in spontaneously hypertensive rats (SHR) was mediated by using a type 8 recombinant adeno-associated virus (rAAV8) vector. Hemodynamics was measured by a Millar Instruments, Inc. (Houston, TX) microtransducer catheter, and atrial natriuretic peptide (ANP) mRNA levels were tested by real-time polymerase chain reaction. Results showed that urinary excretion of 14,15-EET was increased at 2 and 6 months after injection with rAAV-CYP102 F87V and rAAV-CYP2J2 compared with controls (p < 0.05). During the course of the 6-month study, systolic blood pressure significantly decreased in P450 epoxygenase-treated rats, but the CYP2J2-specific inhibitor C26 blocked rAAV-CYP2J2-induced hypotension and the increase in EET production. Cardiac output was improved by P450 epoxygenase expression at 6 months (p < 0.05). Furthermore, cardiac collagen content was reduced in P450 epoxygenase-treated rats. ANP mRNA levels were up-regulated 6- to 14-fold in the myocardium, and ANP expression was significantly increased in both myocardium and plasma in P450 epoxygenase-treated rats. However, epidermal growth factor (EGF) receptor antagonist 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG-1478) significantly attenuated the increase in the EET-induced expression of ANP in vitro. These data indicate that overexpression of P450 epoxygenases attenuates the development of hypertension and improves cardiac function in SHR, and that these effects may be mediated, at least in part, by ANP via activating EGF receptor.

Delivery of recombinant adeno-associated virus-mediated human tissue kallikrein for therapy of chronic renal failure in rats.

The tissue kallikrein-kinin system is important in regulating cardiovascular and renal function, and dysregulation of the system has been implicated in heart and kidney pathologies. These findings suggest that if balance can be restored to the kallikrein-kinin axis, then associated disease progression may be attenuated. To test this hypothesis, recombinant adeno-associated virus (rAAV)-mediated human tissue kallikrein (HK) expression was induced in a rodent model of chronic renal failure involving 5/6 nephrectomy (nephrectomy plus 70% reduction of remaining kidney). rAAV-HK treatment attenuated the rise in blood pressure, glomerular sclerosis, and tubulointerstitial injury observed in this model. rAAV-HK treatment also attenuated renal function decline as measured by urinary microalbumin, osmolarity, and cGMP levels. Reverse transcriptase-polymerase chain reaction analysis showed that rAAV-HK-treated rats had higher levels of bradykinin receptor-2 (B(2)R) and dopamine receptor-1 mRNAs. In contrast, angiotensin II receptor-1, endothelin receptor-A, and vasopressin receptor-2 mRNAs were markedly downregulated in kidneys from HK-treated rats. Bradykinin induced similar changes in receptor levels and prevented transforming growth factor-beta(1)-induced tubulointerstitial fibrosis. The effects of bradykinin could be reversed with the B(2)R antagonist HOE-140. Together, these findings suggest that restoration of the kallikrein-kinin system reduces kidney injury and protects renal function in 5/6-nephrectomized rats via changes in the expression and activation of G protein-coupled receptors including B(2)R.

Male sex hormones exacerbate lung function impairment after bleomycin-induced pulmonary fibrosis.

The roles of sex hormones as modulators of lung function and disease have received significant attention as differential sex responses to various lung insults have been recently reported. The present study used a bleomycin-induced pulmonary fibrosis model in C57BL/6 mice to examine potential sex differences in physiological and pathological outcomes. Endpoints measured included invasive lung function assessment, immunological response, lung collagen deposition, and a quantitative histological analysis of pulmonary fibrosis. Male mice had significantly higher basal static lung compliance than female mice (P < 0.05) and a more pronounced decline in static compliance after bleomycin administration when expressed as overall change or percentage of baseline change (P < 0.05). In contrast, there were no significant differences between the sexes in immune cell infiltration into the lung or in total lung collagen content after bleomycin. Total lung histopathology scores measured using the Ashcroft method did not differ between the sexes, while a quantitative histopathology scoring system designed to determine where within the lung the fibrosis occurred indicated a tendency toward more fibrosis immediately adjacent to airways in bleomycin-treated male versus female mice. Furthermore, castrated male mice exhibited a female-like response to bleomycin while female mice given exogenous androgen exhibited a male-like response. These data indicate that androgens play an exacerbating role in decreased lung function after bleomycin administration, and traditional measures of fibrosis may miss critical differences in lung function between the sexes. Sex differences should be carefully considered when designing and interpreting experimental models of pulmonary fibrosis in mice.

It's all about sex: gender, lung development and lung disease.

Accumulating evidence suggests that gender affects the incidence, susceptibility and severity of several lung diseases. Gender also influences lung development and physiology. Data from both human and animal studies indicate that sex hormones might contribute to disease pathogenesis or serve as protective factors, depending on the disease involved. In this review, the influence of gender and sex hormones on lung development and pathology will be discussed, with specific emphasis on pulmonary fibrosis, asthma and cancer.

Tissue kallikrein reverses insulin resistance and attenuates nephropathy in diabetic rats by activation of phosphatidylinositol 3-kinase/protein kinase B and adenosine 5'-monophosphate-activated protein kinase signaling pathways.

We previously reported that iv delivery of the human tissue kallikrein (HK) gene reduced blood pressure and plasma insulin levels in fructose-induced hypertensive rats with insulin resistance. In the current study, we evaluated the potential of a recombinant adeno-associated viral vector expressing the HK cDNA (rAAV-HK) as a sole, long-term therapy to correct insulin resistance and prevent renal damage in streptozotocin-induced type-2 diabetic rats. Administration of streptozotocin in conjunction with a high-fat diet induced systemic hypertension, diabetes, and renal damage in rats. Delivery of rAAV-HK resulted in a long-term reduction in blood pressure, and fasting plasma insulin was significantly lower in the rAAV-HK group than in the control group. The expression of phosphatidylinositol 3-kinase p110 catalytic subunit and the levels of phosphorylation at residue Thr-308 of Akt, insulin receptor B, and AMP-activated protein kinases were significantly decreased in organs from diabetic animals. These changes were significantly attenuated after rAAV-mediated HK gene therapy. Moreover, rAAV-HK significantly decreased urinary microalbumin excretion, improved creatinine clearance, and increased urinary osmolarity. HK gene therapy also attenuated diabetic renal damage as assessed by histology. Together, these findings demonstrate that rAAV-HK delivery can efficiently attenuate hypertension, insulin resistance, and diabetic nephropathy in streptozotocin-induced diabetic rats.

Phosphorylation of PDZ1 domain attenuates NHERF-1 binding to cellular targets.

NHERF-1 (Na(+)-H(+) exchanger regulatory factor 1, also known as EBP50 ezrin-binding protein of 50 kDa) is a phosphoprotein that assembles multiprotein complexes via two PDZ domains and a C-terminal ezrin-binding domain. Current work utilized metabolic labeling in cultured cells expressing wild type GFP-NHERF-1 to define the physiological importance of NHERF-1 phosphorylation. Treatment of cells with phosphatase inhibitors calyculin A and okadaic acid enhanced NHERF-1 phosphorylation and inhibited its dimerization. Eliminating C-terminal serines abolished the modulation of NHERF-1 dimerization by phosphatase inhibitors and identified the phosphorylation of the PDZ1 domain that attenuated its binding to physiological targets, including beta(2)-adrenergic receptor, platelet-derived growth factor receptor, cystic fibrosis transmembrane conductance regulator, and sodium-phosphate cotransporter type IIa. The major covalent modification of PDZ1 was mapped to serine 77. Confocal microscopy of cultured cells suggested key roles for PDZ1 and ERM-binding domain in localizing NHERF-1 at the cell surface. The substitution S77A eliminated PDZ1 phosphorylation and increased NHERF-1 localization at the cell periphery. In contrast, S77D reduced NHERF-1 colocalization with cortical actin cytoskeleton. These data suggested that serine 77 phosphorylation played key role in modulating NHERF-1 association with plasma membrane targets and identified a novel mechanism by which PDZ1 phosphorylation may transduce hormonal signals to regulate the function of membrane proteins in epithelial tissues.

The impact of sex and sex hormones on lung physiology and disease: lessons from animal studies.

Numerous animal studies have revealed significant effects of sex and sex hormones on normal lung development, lung physiology, and various lung diseases. The primary goal of this review is to summarize knowledge to date on the effects of sex and sex hormones on lung development, physiology, and disease in animals. Specific emphasis will be placed on fibrosis, allergic airway disease, acute lung injury models, respiratory infection, and lung toxicology studies.

Cyclooxygenase-2 deficiency exacerbates bleomycin-induced lung dysfunction but not fibrosis.

Cyclooxygenase (COX)-derived eicosanoids have been implicated in the pathogenesis of pulmonary fibrosis. Uncertainty regarding the influence of COX-2 on experimental pulmonary fibrosis prompted us to clarify the fibrotic and functional effects of intratracheal bleomycin administration in mice genetically deficient in COX-2. Further, the effects of airway-specific COX-1 overexpression on fibrotic and functional outcomes in wild-type and COX-2 knockout mice were assessed. Equivalent increases in airway cell influx, lung collagen content, and histopathologic evidence of fibrosis were observed in wild-type and COX-2 knockout mice 21 d after bleomycin treatment, suggesting that COX-2 deficiency did not alter the extent or severity of fibrosis in this model. However, bleomycin-induced alterations in respiratory mechanics were more severe in COX-2 knockout mice than in wild-type mice, as illustrated by a greater decrease in static compliance compared with genotype-matched, saline-treated control mice (26 +/- 3% versus 11 +/- 4% decreases for COX-2 knockout and wild-type mice, respectively; P < 0.05). The influence of COX-1 overexpression in airway Clara cells was also examined. Whereas the fibrotic effects of bleomycin were not altered in wild-type or COX-2 knockout mice overexpressing COX-1, the exaggerated lung function decrement in bleomycin-treated COX-2 knockout mice was prevented by COX-1 overexpression and coincided with decreased airway cysteinyl leukotriene levels. Collectively, these data suggest an important regulatory role for COX-2 in the maintenance of lung function in the setting of lung fibrosis, but not in the progression of the fibrotic process per se.

Male sex hormones promote vagally mediated reflex airway responsiveness to cholinergic stimulation.

A sex disparity in airway responsiveness to cholinergic stimulation has been observed in laboratory mice in that males are considerably more responsive than females, but the basis for this difference is unclear. In this report, we demonstrate that male sex hormones promote murine airway responsiveness to cholinergic stimulation via vagus nerve-mediated reflex mechanisms. In tissue bath preparations, no sex-based differences were observed in the contractile responses of isolated tracheal and bronchial ring segments to carbachol, indicating that the mechanism(s) responsible for the in vivo sex difference is (are) absent ex vivo. Bilateral cervical vagotomy was found to abolish in vivo airway responsiveness to methacholine in male mice, whereas it did not alter the responses of females, suggesting a regulatory role for male sex hormones in promoting reflex airway constriction. To test this possibility, we next studied mice with altered circulating male sex hormone levels. Castrated male mice displayed airway responsiveness equivalent to that observed in intact females, whereas administration of exogenous testosterone to castrated males restored responsiveness, albeit not to the level observed in intact males. Administration of exogenous testosterone to intact female mice similarly enhanced responsiveness. Importantly, the promotive effects of exogenous testosterone in castrated male and intact female mice were absent when bilateral vagotomy was performed. Together, these data indicate that male sex hormones promote cholinergic airway responsiveness via a vagally mediated reflex mechanism that may be important in the regulation of airway tone in the normal and diseased lung.

Spontaneous airway hyperresponsiveness in estrogen receptor-alpha-deficient mice.

RATIONALE: Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic evidence supports a role for female sex hormones in modulating lung function and airway hyperresponsiveness in humans. OBJECTIVES: To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor-deficient mice. METHODS: Lung function was assessed by a combination of whole-body barometric plethysmography, invasive measurement of airway resistance, and isometric force measurements in isolated bronchial rings. M2 muscarinic receptor expression was assessed by Western blotting, and function was assessed by electrical field stimulation of tracheas in the presence/absence of gallamine. Allergic airway disease was examined after ovalbumin sensitization and exposure. MEASUREMENTS AND MAIN RESULTS: Estrogen receptor-alpha knockout mice exhibit a variety of lung function abnormalities and have enhanced airway responsiveness to inhaled methacholine and serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor expression and function in the lungs. Absence of estrogen receptor-alpha also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge. CONCLUSIONS: These data suggest that estrogen receptor-alpha is a critical regulator of airway hyperresponsiveness in mice.

Regulation of ion transport by the NHERF family of PDZ proteins.

NHERFs are the best-studied apical PDZ proteins that are highly expressed in epithelial cells. Molecular and cellular studies over the past decade show that NHERFs regulate the targeting or trafficking of ion transporters and other membrane proteins and transduce physiological and pathophysiological signals that regulate ion homeostasis in mammals.

The neuronal actin-binding proteins, neurabin I and neurabin II, recruit specific isoforms of protein phosphatase-1 catalytic subunits.

Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1gamma1 and PP1alpha but not PP1beta. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1gamma1 and PP1alpha from brain extracts and associated poorly with PP1beta. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1beta and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1gamma1 > PP1alpha > PP1beta. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1gamma null mice. In the absence of PP1gamma1, both neurabins showed enhanced association with PP1alpha but not PP1beta. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1gamma1 and PP1alpha to the mammalian synapse.