Linda Sanders

Estrogen restores cognition and cholinergic phenotype in an animal model of Down syndrome

Estrogen maintains normal function of basal forebrain (BF) cholinergic neurons and estrogen replacement therapy (ERT) has therefore been proposed as a therapy for Alzheimers disease (AD). We provide evidence to support this hypothesis in an animal model of Down syndrome (DS), a chromosome 16 segmental trisomy (Ts65Dn) mouse. These mice develop cholinergic degeneration similar to young adults with DS and AD patients. ERT has not been tested in women with DS, even though they are more likely than normosomic women to develop early menopause and AD. Female Ts65Dn and normosomic mice (11-15 months) received a subcutaneous estrogen pellet or a sham operation. After 60 days, estrogen treatment improved learning of a T-maze task and normalized behavior in the Ts65Dn mice in reversal learning of the task, a measure of cognitive flexibility. Stereological evaluation of choline acetyltransferase (ChAT) immunopositive BF neurons showed that estrogen increased cell size and total number of cholinergic neurons in the medial septum of Ts65Dn mice. In addition, estrogen increased NGF protein levels in the BF of trisomic mice. These findings support the emerging hypothesis that estrogen may play a protective role during neurodegeneration and cognitive decline, particularly in cholinergic BF neuronal systems underlying cognition. The findings also indicate that estrogen may act, at least partially, via endogenous growth factors. Collectively, the data suggest that ERT may be a viable therapeutic approach for women with DS coupled with dementia.

Transforming Growth Factor-β Signaling Promotes Pulmonary Hypertension Caused by Schistosoma Mansoni

Background— The pathogenic mechanisms underlying pulmonary arterial hypertension resulting from schistosomiasis, one of the most common causes of pulmonary hypertension worldwide, remain unknown. We hypothesized that transforming growth factor-&bgr; (TGF-&bgr;) signaling as a consequence of Th2 inflammation is critical for the pathogenesis of this disease. Methods and Results— Mice sensitized and subsequently challenged with Schistosoma mansoni eggs developed pulmonary hypertension associated with an increase in right ventricular systolic pressure, thickening of the pulmonary artery media, and right ventricular hypertrophy. Rho-kinase–dependent vasoconstriction accounted for ≈60% of the increase in right ventricular systolic pressure. The pulmonary vascular remodeling and pulmonary hypertension were dependent on increased TGF-&bgr; signaling, as pharmacological blockade of the TGF-&bgr; ligand and receptor, and mice lacking Smad3 were significantly protected from Schistosoma-induced pulmonary hypertension. Blockade of TGF-&bgr; signaling also led to a decrease in interleukin-4 and interleukin-13 concentrations, which drive the Th2 responses characteristic of schistosomiasis lung pathology. Lungs of patients with schistosomiasis-associated pulmonary arterial hypertension have evidence of TGF-&bgr; signaling in their remodeled pulmonary arteries. Conclusion— Experimental S mansoni–induced pulmonary vascular disease relies on canonical TGF-&bgr; signaling.

TGF-β Signaling Promotes Pulmonary Hypertension Caused by Schistosoma Mansoni

Background— The pathogenic mechanisms underlying pulmonary arterial hypertension resulting from schistosomiasis, one of the most common causes of pulmonary hypertension worldwide, remain unknown. We hypothesized that transforming growth factor-&bgr; (TGF-&bgr;) signaling as a consequence of Th2 inflammation is critical for the pathogenesis of this disease. Methods and Results— Mice sensitized and subsequently challenged with Schistosoma mansoni eggs developed pulmonary hypertension associated with an increase in right ventricular systolic pressure, thickening of the pulmonary artery media, and right ventricular hypertrophy. Rho-kinase–dependent vasoconstriction accounted for ≈60% of the increase in right ventricular systolic pressure. The pulmonary vascular remodeling and pulmonary hypertension were dependent on increased TGF-&bgr; signaling, as pharmacological blockade of the TGF-&bgr; ligand and receptor, and mice lacking Smad3 were significantly protected from Schistosoma-induced pulmonary hypertension. Blockade of TGF-&bgr; signaling also led to a decrease in interleukin-4 and interleukin-13 concentrations, which drive the Th2 responses characteristic of schistosomiasis lung pathology. Lungs of patients with schistosomiasis-associated pulmonary arterial hypertension have evidence of TGF-&bgr; signaling in their remodeled pulmonary arteries. Conclusion— Experimental S mansoni–induced pulmonary vascular disease relies on canonical TGF-&bgr; signaling.

The Causal Role of IL-4 and IL-13 in Schistosoma mansoni Pulmonary Hypertension

RATIONALE The etiology of schistosomiasis-associated pulmonary arterial hypertension (PAH), a major cause of PAH worldwide, is poorly understood. Schistosoma mansoni exposure results in prototypical type-2 inflammation. Furthermore, transforming growth factor (TGF)-β signaling is required for experimental pulmonary hypertension (PH) caused by Schistosoma exposure. OBJECTIVES We hypothesized type-2 inflammation driven by IL-4 and IL-13 is necessary for Schistosoma-induced TGF-β-dependent vascular remodeling. METHODS Wild-type, IL-4(-/-), IL-13(-/-), and IL-4(-/-)IL-13(-/-) mice (C57BL6/J background) were intraperitoneally sensitized and intravenously challenged with S. mansoni eggs to induce experimental PH. Right ventricular catheterization was then performed, followed by quantitative analysis of the lung tissue. Lung tissue from patients with schistosomiasis-associated and connective tissue disease-associated PAH was also systematically analyzed. MEASUREMENTS AND MAIN RESULTS Mice with experimental Schistosoma-induced PH had evidence of increased IL-4 and IL-13 signaling. IL-4(-/-)IL-13(-/-) mice, but not single knockout IL-4(-/-) or IL-13(-/-) mice, were protected from Schistosoma-induced PH, with decreased right ventricular pressures, pulmonary vascular remodeling, and right ventricular hypertrophy. IL-4(-/-)IL-13(-/-) mice had less pulmonary vascular phospho-signal transducer and activator of transcription 6 (STAT6) and phospho-Smad2/3 activity, potentially caused by decreased TGF-β activation by macrophages. In vivo treatment with a STAT6 inhibitor and IL-4(-/-)IL-13(-/-) bone marrow transplantation also protected against Schistosoma-PH. Lung tissue from patients with schistosomiasis-associated and connective tissue disease-associated PAH had evidence of type-2 inflammation. CONCLUSIONS Combined IL-4 and IL-13 deficiency is required for protection against TGF-β-induced pulmonary vascular disease after Schistosoma exposure, and targeted inhibition of this pathway is a potential novel therapeutic approach for patients with schistosomiasis-associated PAH.

Protein microarray analysis of nasal polyps from aspirin-sensitive and aspirin-tolerant patients with chronic rhinosinusitis

Background The purpose of this study was to apply protein microarray technology to the study of sinonasal tissue and to identify differential protein expression in nasal polyps from aspirin-sensitive (AS) versus aspirin-tolerant (AT) patients with chronic rhinosinusitis (CRS) and CRS with nasal polyps (CRSwNPs). Methods Nasal polyp specimens were prospectively obtained from two groups of patients with CRSwNP. The test group (AS) consisted of five patients that were diagnosed with CRSwNP and intolerance to aspirin based on medical history and physical exam. The control group (AT) consisted of four AT patients with CRSwNP. Protein was extracted and labeled from harvested polyps and the Sigma Panorama Antibody Microarray–Cell Signaling Kit was used to identify differences in protein expression between the two polyp groups. Western blot analysis was used to validate the results of the protein microarray. Results The protein microarray showed a greater than twofold change in expression of both beta-adaptin and heat shock protein 70 (HSP70). Western blot analysis confirmed up-regulation of beta-adaptin and HSP70 in nasal polyp tissue from AS patients. Conclusion Pooled samples of AS and AT nasal polyps evaluated by protein microarray show distinct protein expression profiles in the stress response and receptor-mediated endocytosis pathways. This study establishes the successful application of protein microarray technology to study nasal polyposis, which in turn can be validated by Western blot analysis.

Severe pulmonary hypertension is associated with altered right ventricle metabolic substrate uptake

In severe pulmonary hypertension (SPH), prior studies have shown an increase in right ventricle (RV) uptake of glucose, but it is unclear whether there is a change in the relative utilization of fatty acids. We hypothesized that in the RV in SPH, as in left ventricular (LV) failure, there is altered substrate utilization, with increased glucose uptake and decreased fatty acid uptake. SPH was induced in rats by treatment with the VEGF receptor inhibitor SU5416 and 3 wk of hypoxia (10% FiO2 ), followed by an additional 4 wk of normoxia (SU-Hx group). Control rats were treated with carboxymethylcellulose vehicle and 7 wk of normoxia (CMC-Nx group). The rodents then underwent positron emission tomography with sequential administration of two radiotracers, 2-deoxy-2-[(18)F]fluoroglucose ((18)F-FDG) and 14-(R,S)-[(18)F]fluoro-6-thia-heptadecanoic acid ((18)F-FTHA), analogs of glucose and fatty acid, respectively. Five CMC-Nx and 3 SU-Hx rats completed the entire experimental protocol. In the RV, there was a mild increase in (18)F-FDG uptake (1.35-fold, P = 0.085) and a significant decrease in (18)F-FTHA uptake (-2.1-fold, P < 0.05) in the SU-Hx rats relative to the CMC-Nx rats. In the LV, SU-Hx rats had less uptake of both radiotracers compared with CMC-Nx rats. Less RV fatty acid uptake in SPH was corroborated by decreased fatty acid transporters and enzymes in the RV tissue, and specifically a decrease in lipoprotein lipase. In the RV in rats with SPH, there is a major shift in metabolic substrate preference, largely due to decreased fatty acid uptake.

TGF-β activation by bone marrow-derived thrombospondin-1 causes Schistosoma - and hypoxia-induced pulmonary hypertension

Pulmonary arterial hypertension (PAH) is an obstructive disease of the precapillary pulmonary arteries. Schistosomiasis-associated PAH shares altered vascular TGF-β signalling with idiopathic, heritable and autoimmune-associated etiologies; moreover, TGF-β blockade can prevent experimental pulmonary hypertension (PH) in pre-clinical models. TGF-β is regulated at the level of activation, but how TGF-β is activated in this disease is unknown. Here we show TGF-β activation by thrombospondin-1 (TSP-1) is both required and sufficient for the development of PH in Schistosoma-exposed mice. Following Schistosoma exposure, TSP-1 levels in the lung increase, via recruitment of circulating monocytes, while TSP-1 inhibition or knockout bone marrow prevents TGF-β activation and protects against PH development. TSP-1 blockade also prevents the PH in a second model, chronic hypoxia. Lastly, the plasma concentration of TSP-1 is significantly increased in subjects with scleroderma following PAH development. Targeting TSP-1-dependent activation of TGF-β could thus be a therapeutic approach in TGF-β-dependent vascular diseases.

Novosphingobium and Its Potential Role in Chronic Obstructive Pulmonary Diseases: Insights from Microbiome Studies

Bacterial infection of lung airways underlies some of the main complications of COPD, significantly impacting disease progression and outcome. Colonization by bacteria may further synergize, amplify, or trigger pathways of tissue damage started by cigarette smoke, contributing to the characteristic airway inflammation and alveolar destruction of COPD. We sought to elucidate the presence and types of lung bacterial populations in different stages of COPD, aimed at revealing important insights into the pathobiology of the disease. Sequencing of the bacterial small subunit ribosomal RNA gene in 55 well-characterized clinical lung samples, revealed the presence of Novosphingobium spp. (>2% abundance) in lungs of patients with GOLD 3-GOLD 4 COPD, cystic fibrosis and a subset of control individuals. Novosphingobium-specific quantitative PCR was concordant with the sequence data and high levels of Novosphingobium spp. were quantifiable in advanced COPD, but not from other disease stages. Using a mouse model of subacute lung injury due to inhalation of cigarette smoke, bronchoalveolar lavage neutrophil and macrophage counts were significantly higher in mice challenged intratracheally with N. panipatense compared to control mice (p<0.01). Frequencies of neutrophils and macrophages in lung tissue were increased in mice challenged with N. panipatense at room air compared to controls. However, we did not observe an interaction between N. panipatense and subacute cigarette smoke exposure in the mouse. In conclusion, Novosphingobium spp. are present in more severe COPD disease, and increase inflammation in a mouse model of smoke exposure.

Rtp801 suppression of epithelial mTORC1 augments endotoxin-induced lung inflammation.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular responses to environmental stress. mTOR (and its primary complex mTORC1) is, therefore, ideally positioned to regulate lung inflammatory responses to an environmental insult, a function directly relevant to disease states such as the acute respiratory distress syndrome. Our previous work in cigarette smoke-induced emphysema identified a novel protective role of pulmonary mTORC1 signaling. However, studies of the impact of mTORC1 on the development of acute lung injury are conflicting. We hypothesized that Rtp801, an endogenous inhibitor of mTORC1, which is predominantly expressed in alveolar type II epithelial cells, is activated during endotoxin-induced lung injury and functions to suppress anti-inflammatory epithelial mTORC1 responses. We administered intratracheal lipopolysaccharide to wild-type mice and observed a significant increase in lung Rtp801 mRNA. In lipopolysaccharide-treated Rtp801(-/-) mice, epithelial mTORC1 activation significantly increased and was associated with an attenuation of lung inflammation. We reversed the anti-inflammatory phenotype of Rtp801(-/-) mice with the mTORC1 inhibitor, rapamycin, reassuring against mTORC1-independent effects of Rtp801. We confirmed the proinflammatory effects of Rtp801 by generating a transgenic Rtp801 overexpressing mouse, which displayed augmented inflammatory responses to intratracheal endotoxin. These data suggest that epithelial mTORC1 activity plays a protective role against lung injury, and its inhibition by Rtp801 exacerbates alveolar injury caused by endotoxin.

Circulating RNA Transcripts Identify Therapeutic Response in Cystic Fibrosis Lung Disease

RATIONALE Circulating leukocyte RNA transcripts are systemic markers of inflammation, which have not been studied in cystic fibrosis (CF) lung disease. Although the standard assessment of pulmonary treatment response is FEV(1), a measure of airflow limitation, the lack of systemic markers to reflect changes in lung inflammation critically limits the testing of proposed therapeutics. OBJECTIVES We sought to prospectively identify and validate peripheral blood leukocyte genes that could mark resolution of pulmonary infection and inflammation using a model by which RNA transcripts could increase the predictive value of spirometry. METHODS Peripheral blood mononuclear cells were isolated from 10 patients with CF and acute pulmonary exacerbations before and after therapy. RNA expression profiling revealed that 10 genes significantly changed with treatment when compared with matched non-CF and control subjects with stable CF to establish baseline transcript abundance. Peripheral blood mononuclear cell RNA transcripts were prospectively validated, using real-time polymerase chain reaction amplification, in an independent cohort of acutely ill patients with CF (n = 14). Patients who responded to therapy were analyzed using general estimating equations and multiple logistic regression, such that changes in FEV(1)% predicted were regressed with transcript changes. MEASUREMENTS AND MAIN RESULTS Three genes, CD64, ADAM9, and CD36, were significant and independent predictors of a therapeutic response beyond that of FEV(1) alone (P < 0.05). In both cohorts, receiver operating characteristic analysis revealed greater accuracy when genes were combined with FEV(1). CONCLUSIONS Circulating mononuclear cell transcripts characterize a response to the treatment of pulmonary exacerbations. Even in small patient cohorts, changes in gene expression in conjunction with FEV(1) may enhance current outcomes measures for treatment response.