Role Of Smooth Muscle Progenitor Cells In Asthmatic Airway Remodeling

Background:Asthma is a chronic inflammatory disease characterized by reversible airflow limitation and airway hyperresponsiveness. It remains a severe health problem since current therapies are directed to suppressing, rather than preventing or reversing, the primary disease process.Persistent inflammation in airway tissues may lead to structural changes known as airway remodeling and consequently airway obstruction that is not fully reversible and progressive loss of lung function over time. Airway remodeling in asthma is a complex process that involves structural changes in virtually all tissues of the airway wall. Airway remodeling, a central feature of asthma, is characterized by airway smooth muscle hypertrophy and hyperplasia, collagen deposition to sub-epithelial basement membrane, hyperplasia of goblet cells, thickening of airway mucosa and an increase in vascularity. Current treatments may indirectly control airway remodeling through a reduction of inflammation but such a kind of approach is only in part successful. Airway smooth muscle plays a multifaceted role in the pathogenesis of airway remodeling in asthma,including airway myocytes to airway inflammation, airway wall remodeling, and airflow obstruction in this prevalent disease syndrome. Together, these roles make airway smooth muscle an attractive target for asthma therapy.Recent animal data suggest that vascular smooth muscle cells within the neointima of the vessel wall may originate from bone marrow, providing indirect evidence for circulating smooth muscle progenitor cells (SPCs).The location, mobilization, and function of such progenitors is still poorly understood, but may include reparative or alternatively proliferative consequences for vascular injury development and progression. These observations provide a platform for re-evaluation of smooth muscle cell heterogeneity and progenitor biology in asthma, with implications for therapy of airway remodeling in asthma.Previous studies have suggested that the number of CD34+ progenitor cells is elevated in the peripheral blood of asthmatic patients and PDGF has a potential pathological role in asthmatic airway remodeling. Both of them are correlated with smooth muscle progenitor cells, one is an important characteristic, another is differentiation inductor. It means smooth muscle progenitor cells probably participate in asthmatic airway remodeling. Clearly, a greater understanding of the pathogenesis of smooth muscle progenitor cell in asthmatic airway remodeling is critical to the development of better therapeutic modalities.In the present study, we tested the hypothesis that smooth muscle progenitor cells may play an important role in asthmatic airway remodeling.Part I Influence of atomization inhaled ovalbumin on smooth muscle progenitor cells in peripheral blood from rat with chronic asthma.Objective:To investigate the influence of atomization inhaled ovalbumin on smooth muscle progenitor cells in peripheral blood from rat with chronic asthma.Methods and materials: Ten Wistar rats were randomly divided into two groups. OVA sensitization was performed by concurrent subcutaneous injection of OVA adsorbed to aluminum hydroxide.Fouteen days later, the animals were challenged by aerosol with an OVA solution or with phosphate-buffered saline for 8 weeks. All animals were killed 24hours after antigen challenge. On killing, lungs of the animals were rinsed with saline and total and differential cell counts were performed. The peripheral blood mononuclear cells (MNCs) were isolated from the peripheral blood of rats by density gradient centrifugation with Ficoll.Examination expressions of CD34 and SM-MHC in asthma group and control group by flow cytometric analysis.Result:After8 weeks of OVA challenge, inflammatory response and structural changes were found in airway. The area of smooth muscle in the large airway in asthmatic groups was significantly increased as compared with that in control groups. Compared with those in control group, lymphocytes, neutrophils, eosinophils, goblet cells were increased. The area of airway smooth muscle was significantly increased mainly in the large airways of rats after challenged to OVA for 8 weeks. The expressions of CD34 and SM-MHC in asthma group were higher than control group. Conclusion:Atomization inhaled ovalbumin can increase smooth muscle progenitor cells in peripheral blood from rat with chronic asthma. The increased expression of smooth muscle progenitor cells suggested that they may play a important role in airway remodeling of asthma.Part II Isolation, culture and characterization of bone marrow mesenchymal stem cells, and induce mMSCs differentiate into smooth muscle progenitor cellsObjective:To explore the potential of rat bone marrow mesenchymal stem cells (MSCs) to differentiate into smooth muscle progenitor cells.Methods and materials:After the tibias and femurs were dissected from 4～6 week-old rats ,BM mononuclear cells(MNCs)were plated in the 75cm² flasks, at a concentration of 1 10⁶/cm², in 2ml DMEM-LG medium supplemented with 10%FBS, 4 mM L-glutamine, 100u/ml penicillin and 100,ug/ml streptomycin. Flasks were maintained in incubator with 5%CO₂ at 37.0℃. After 24h, nonadherent cells were eliminated by medium changing, and then the medium were changed every 3 days. The cells were grown for 2-3 weeks until attaching to 80% confluences, and then they were trypsinized with 0.05% trypsin-0.02% EDTA and replanted at about 10 cells/well in the 96-well flasks. When the colonies were grown up, cells were expanded at density vary from 1.5 103/cm² to 3.0 103/cm². The characters of the cell, such as morphology, cell growth curve, cell cycle and phenotype were demonstrated. Their abilities to differentiate along adipocytic and osteoblastic pathways were also investigated.The MSCs, which were obtained, cultured in DMEM medium containing 20 %FBS, 50ng/ ml Platelet-derived growth factor - BB (PDGF - BB). The cultured cells in vitro were identified with smooth muscle cell specificαactin (α-SMA) and CD34 by immunohistochemistry and flow cytometric analysis was used to identify the smooth muscle progenitor cells level after PDGF - BB effect.Result: The MSCs population consisted of spindle- and star-shaped cells.Flow-cytometric analysis showed that they were have typical characteristics.MSCs can be differentiated into adipocyte and osteoblast cell.Smooth muscle-like cells outgrew from the culture of MSCs in presence of PDGF– BB after cultured 2 weeks. These cells were positive stain forα-SMA and CD34, butα-SMA and CD34 were not expressed in fresh isolated MSCs.Conclusion:MSCs have been isolated and purified successfully.The growing cell population consisted of spindle- and star-shaped cells with significant renewal capacity and multiple differentiaed abilities as they were culture.Smooth muscle progenitor cells can outgrow from bone MSC, This method may be useful in study of smooth muscle progenitor cell.Part III Role of smooth muscle progenitor cells in airway remodeling in asthmaObjective:To explore the role of smooth muscle progenitor cells in airway remodeling in asthma.Methods and materials:Twenty Wistar rats were randomly divided into four groups: female control+SPC group, female asthma group, female asthma+SPC group, male asthma group. OVA sensitization was performed by concurrent subcutaneous injection of OVA adsorbed to aluminum hydroxide.Fouteen days later, the animals were challenged by aerosol with an OVA solution or with phosphate-buffered saline for 8 weeks.Smooth progenitor cells(1.0×10⁶) which isolated from male rats was intravenous injection since fourth week,the injections were repeated for four times once a week. All animals were killed 24hours after antiger challenge. Airway samples were prepared for histology by formalin fixation and paraffin embedding. Each groups rats airway smooth muscle cells (ASMC) were isolated and subculture. Airway samples and ASMC were examinated Sry (sex-determining region on the Y chromosome) positive cell count by hybridization in situ(ISH).Result:Both of airway samples and ASMC in female asthma+SPC group were examinnted Sry positive cell count by hybridization in situ(ISH).But female control+SPC group were negative results.Conclusion:Smooth muscle progenitor cells take participate in asthmatic airway remodeling and play a critical role. Part IV Effect of atomization inhaled Sirolimus to smooth muscle progenitor cells in airway remodeling of asthma.Objective:To investigate the effect of sirolimus on proliferation and differentiation of mooth muscle progenitor cells and effect of atomization inhaled Sirolimus to smooth muscle progenitor cells in airway remodeling of asthma.Methods and materials:The bone marrow mesenchymal cells (MSCs) were isolated from the bone marrow of rats by adhering to the culture plastic.MSCs were cultured infibronectin-coated dishes in smooth muscle progenitor cells growth supplements with or without sirolimus (final concentrations: 0.1, 1, 10, 100ng/ml) for 3 days, SPCs proliferation were assayed by MTT assay for various time points (0h, 12 h, 24 h, 48 h). After 10 days primarily cultured, attached cells were treated with sirolimus(final concentrations: 0.1, 1ng/ml) , SPCs were identified as adherent cells positive for a -SMA by indirect immunohistochemistry staining. Fifteen Wistar rats were randomly divided into three groups,female control group, female OVA+SPC group and female OVA/sirolimus+SPC group. OVA sensitization was performed by concurrent subcutaneous injection of OVA adsorbed to aluminum hydroxide.Fouteen days later, the animals were challenged by aerosol with an OVA solution or with phosphate-buffered saline for 8 weeks.The female OVA/sirolimus+SPC group also give atomization inhaled Sirolimus for 10 mins after OVA challenge. Airway samples were prepared for histology by formalin fixation and paraffin embedding. Each groups rats airway smooth muscle cells (ASMC) were isolated and subculture. Airway samples were examinated Sry positive cell count by hybridization in situ(ISH).Result:Sirolimus also significantly inhibited the proliferative of SPC in a time and dose dependent manner.After cultured in different drug concentration of sirolimus of DM,0.1ng/ml,1ng/ml,10ng/ml and 100ng/ml for 48 hours,the optical density of each groups were 0.75±0.06,0.44±0.04,0.33±0.02,0.20±0.01 and 0.18±0.01(P<0.05).SPC number diferentiated from MSC (final concentrations:DM, 0.1, 1ng/ml) at 10 days was significantly lower in sirolimus treated cells in a dose—dependent manner than that of vehicle treated cells.After cultured in different drug concentration of sirolimus of DM,0.1ng/ml and 1ng/ml for 10 days, the rate of a-SMA-positive SPCs from MSCs of each groups were 71%,24% and 4%(P <0.05). Both of OVA/OVA and OVA/sirolimus groups rats'airway samples were examinnted Sry positive cells by hybridization in situ(ISH).But Sry positive cell count in OVA/sirolimus group was lower than OVA/OVA group. OVA/PBS control+SPC group were negative results.Conclusion:Sirolimus could inhibitthe proliferation and diferentiation of smooth muscle progenitor cells,and it helpful for therapy of asthmatic airway remodeling by reduce smooth muscle progenitor cells take participate in it.