Effects of experimental asthma on inflammation and lung mechanics in sickle cell mice.

Experimental asthma increases eosinophil and collagen deposition in the lungs of sickle cell disease (SCD) mice to a greater extent than in control mice. However, the effects of asthma on inflammation and airway physiology remain unclear. To determine effects of asthma on pulmonary inflammation and airway mechanics in SCD mice, hematopoietic stem cell transplantation was used to generate chimeric SCD and hemoglobin A mice. Experimental asthma was induced by sensitizing mice with ovalbumin (OVA). Airway mechanics were assessed using forced oscillation techniques. Mouse lungs were examined histologically and physiologically. Cytokine, chemokine, and growth factors in bronchoalveolar lavage fluid were determined by multiplex. IgE was quantified by ELISA. LDH was quantified using a colorimetric enzymatic assay. At baseline (nonsensitized), chimeric SCD mice developed hemolytic anemia with sickled red blood cells, mild leukocytosis, and increased vascular endothelial growth factor and IL-13 compared with chimeric hemoglobin A mice. Experimental asthma increased perialveolar eosinophils, plasma IgE, and bronchoalveolar lavage fluid IL-1ß, IL-4, IL-6, and monocyte chemotactic protein 1 in chimeric hemoglobin A and SCD mice. IFN-γ levels were reduced in both groups. IL-5 was preferentially increased in chimeric SCD mice but not in hemoglobin A mice. Positive end-expiratory pressures and methacholine studies revealed that chimeric SCD mice had greater resistance in large and small airways compared with hemoglobin A mice at baseline and after OVA sensitization. SCD alone induces a baseline lung pathology that increases large and small airway resistance and primes the lungs to increased inflammation and airway hyperresponsiveness after OVA sensitization.

Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in a murine model of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by oxidative stress, impaired vascular function, and attenuated angiogenesis. The tight-skin (Tsk(-/+)) mouse is a model of SSc that displays many of the cellular features of the clinical disease. We tested the hypotheses that abnormal fibrillin-1 expression and chronic phospholipid oxidation occur in Tsk(-/+) mice and, furthermore, that these factors precipitate a prooxidant state, collagen-related protein expression, apoptosis, and mesenchymal transition in endothelial cells cultured on Tsk(-/+) extracellular matrix. Human umbilical vein endothelial cells were seeded on microfibrils isolated from skin of C57BL/6J (control) and Tsk(-/+) mice in the presence or absence of chronic pretreatment with the apolipoprotein Apo A-I mimetic D-4F (1 mg·kg(-1)·day(-1) ip for 6 to 8 wk). Nitric oxide-to-superoxide anion ratio was assessed 12 h after culture, and cell proliferation, apoptosis, and phenotype were studied 72 h after culture. Tsk(-/+) mice demonstrated abnormal "big fibrillin" expression (405 kDa) by Western blot analysis compared with control. Endothelial cells cultured on microfibrils prepared from Tsk(-/+) mice demonstrated reduced proliferation, a prooxidant state (reduced nitric oxide-to-superoxide anion ratio), increased apoptosis, and collagen-related protein expression associated with mesenchymal transition. Chronic D-4F pretreatment of Tsk(-/+) mice attenuated many of these adverse effects. The findings demonstrate that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in Tsk(-/+) mice. This mesenchymal transition may contribute to the reduction in angiogenesis that is known to occur in this model of SSc.

Genetic deletion of apolipoprotein A-I increases airway hyperresponsiveness, inflammation, and collagen deposition in the lung.

The relationship between high-density lipoprotein and pulmonary function is unclear. To determine mechanistic relationships we investigated the effects of genetic deletion of apolipoprotein A-I (apoA-I) on plasma lipids, paraoxonase (PON1), pro-inflammatory HDL (p-HDL), vasodilatation, airway hyperresponsiveness and pulmonary oxidative stress, and inflammation. ApoA-I null (apoA-I(-/-)) mice had reduced total and HDL cholesterol but increased pro-inflammatory HDL compared with C57BL/6J mice. Although PON1 protein was increased in apoA-I(-/-) mice, PON1 activity was decreased. ApoA-I deficiency did not alter vasodilatation of facialis arteries, but it did alter relaxation responses of pulmonary arteries. Central airway resistance was unaltered. However, airway resistance mediated by tissue dampening and elastance were increased in apoA-I(-/-) mice, a finding also confirmed by positive end-expiratory pressure (PEEP) studies. Inflammatory cells, collagen deposition, 3-nitrotyrosine, and 4-hydroxy-2-nonenal were increased in apoA-I(-/-) lungs but not oxidized phospholipids. Colocalization of 4-hydroxy-2-nonenal with transforming growth factor beta-1 (TGFbeta-1 was increased in apoA-I(-/-) lungs. Xanthine oxidase, myeloperoxidase and endothelial nitric oxide synthase were increased in apoA-I(-/-) lungs. Dichlorodihydrofluorescein-detectable oxidants were increased in bronchoalveolar lavage fluid (BALF) in apoA-I(-/-) mice. In contrast, BALF nitrite+nitrate levels were decreased in apoA-I(-/-) mice. These data demonstrate that apoA-I plays important roles in limiting pulmonary inflammation and oxidative stress, which if not prevented, will decrease pulmonary artery vasodilatation and increase airway hyperresponsiveness.

Histopathology of experimentally induced asthma in a murine model of sickle cell disease.

Asthma is a comorbid condition associated with increased rates of pain, acute chest syndrome, and premature death in human sickle cell disease (SCD). We developed an experimental asthma model in SCD and control mice expressing either normal human or murine hemoglobin to determine its effect on mortality and lung pathology. To induce lung inflammation, experimental mice were sensitized to ovalbumin (OVA) by subcutaneous OVA implantation (Sen), allowed 2 weeks to recover, and then divided into 2 groups, each receiving over a subsequent 10-day period the same dosage of aerosolized OVA but 2 different levels of exposure: 15 minutes (LoSen) and 30 minutes (HiSen). During recovery, 10% of SCD mice died compared with no deaths in control mice. An additional 30% of HiSen SCD mice died during aerosolization compared with 10% in LoSen SCD. Histologic indices of lung inflammation (eg, eosinophil recruitment, airway and vessel wall thickening, and immunoreactive TGFbeta and fsp-1) and bronchial alveolar lavage fluid eosinophil peroxidase activity differentially increased in sensitized mice compared with unsensitized mice. Our findings indicate SCD mice with experimentally induced asthma are more susceptible to death and pulmonary inflammation compared with control mice, suggesting that asthma contributes significantly to morbidity and mortality in SCD.

The use of sterically stabilized liposomes to treat asthma.

Asthma is characterized by airway hyperresponsiveness, chronic inflammation, and airway remodeling, which may lead to progressive, irreversible lung damage. Liposomes have been used for the delivery of aerosolized asthma medications into the lungs. This method could facilitate sustained action of steroids while using only a fraction of the dosage and a less frequent dosing interval than conventional therapy. We describe the evaluation of the effect of budesonide encapsulated in sterically stabilized liposomes on lung inflammation and airway hyperreactivity in a mouse model of asthma. We outline the determination of markers implicated in the progression of asthma, including histopathology, eosinophil peroxidase activity in bronchoalveolar lavage, and airway hyperresponsiveness to methacholine. Weekly administration of budesonide in sterically stabilized liposomes results in a significant reduction in the total lung inflammation score, peripheral blood eosinophil counts, and the total serum IgE level, similar to that obtained with daily budesonide. Airway hyperresponsiveness to methacholine challenge decreases significantly in the group treated with weekly budesonide in sterically stabilized liposomes, while it does not decrease in the daily budesonide group.

Transient repetitive exposure to low level light therapy enhances collateral blood vessel growth in the ischemic hindlimb of the tight skin mouse.

The tight skin mouse (Tsk(-/+)) is a model of scleroderma characterized by impaired vasoreactivity, increased oxidative stress, attenuated angiogenic response to VEGF and production of the angiogenesis inhibitor angiostatin. Low-level light therapy (LLLT) stimulates angiogenesis in myocardial infarction and chemotherapy-induced mucositis. We hypothesize that repetitive LLLT restores vessel growth in the ischemic hindlimb of Tsk(-/+) mice by attenuating angiostatin and enhancing angiomotin effects in vivo. C57Bl/6J and Tsk(-/+) mice underwent ligation of the femoral artery. Relative blood flow to the foot was measured using a laser Doppler imager. Tsk(-/+) mice received LLLT (670 nm, 50 mW cm(-2), 30 J cm(-2)) for 10 min per day for 14 days. Vascular density was determined using lycopersicom lectin staining. Immunofluorescent labeling, Western blot analysis and immunoprecipitation were used to determine angiostatin and angiomotin expression. Recovery of blood flow to the ischemic limb was reduced in Tsk(-/+) compared with C57Bl/6 mice 2 weeks after surgery. LLLT treatment of Tsk(-/+) mice restored blood flow to levels observed in C57Bl/6 mice. Vascular density was decreased, angiostatin expression was enhanced and angiomotin depressed in the ischemic hindlimb of Tsk(-/+) mice. LLLT treatment reversed these abnormalities. LLLT stimulates angiogenesis by increasing angiomotin and decreasing angiostatin expression in the ischemic hindlimb of Tsk(-/+) mice.

Efficacy of liposomal budesonide in experimental asthma.

BACKGROUND: Inhaled corticosteroids, such as budesonide, attenuate the inflammatory response in asthma. However, patient noncompliance and side effects of available inhaled corticosteroids limit their use. Liposomes are currently used in medicine to deliver a variety of drugs. OBJECTIVE: The objective of our study was to determine whether weekly therapy with budesonide encapsulated in sterically stabilized (stealth) liposomes would be comparable to daily budesonide therapy in reducing allergic inflammation. METHODS: Ovalbumin-sensitized C57/Black 6 mice received aerosolized (1) budesonide encapsulated in stealth or conventional liposomes, administered weekly, (2) budesonide (without liposomes), administered either daily or weekly, or (3) empty stealth liposomes, administered weekly. All treatment groups were compared with sensitized untreated or unsensitized mice. Histopathologic examination of the lung tissues and measurements of eosinophil peroxidase activity, peripheral blood eosinophil counts, and total serum IgE levels were done weekly for 4 weeks. RESULTS: Weekly therapy with budesonide encapsulated in stealth liposomes was as effective as daily budesonide therapy in decreasing lung inflammation and lowering eosinophil peroxidase activity, peripheral blood eosinophils, and total serum IgE levels. In none of the other groups was there a significant decrease in the inflammatory parameters evaluated. CONCLUSION: We conclude that weekly therapy with budesonide encapsulated in stealth liposomes is as effective as daily budesonide in reducing markers of lung inflammation in experimental asthma. This novel strategy offers an effective alternative to standard daily budesonide therapy in asthma and has the potential to reduce toxicity and improve compliance.