The Study Of Bone Marrow Mesenchymal Stem Cells Transplantation For Treatment Of Pulmonary Artery Hypertension

Objective:To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) transplantation on the model of pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT) and discuss the machenism.Methods:1. Pulmonary arterial hypertension model establishment and evaluationMCT was prepared as described and was dissolved in1N HC1, neutralized to pH7.4with0.5N NaOH, and diluted with saline just before injection. Male S-D rats were randomly assigned to2groups (n=10) as follows:PAH group:animals that received a abdominal injection of50mg/kg MCT; control group:animals that received a abdominal injection of0.9%saline instead of MCT. One week after MCT injection, the rats were anesthetized and inserted with a3F-Miller microtip catheter via the right jugular vein into the right ventricle to obtain base line measurements of hemodynamics such as right ventricular systolic pressure (RVSP), mean right ventricular pressure (MRVP), and mean pulmonary arterial pressure (MPAP). The rats were euthanized after hemodynamic measurements, the lung and heart were quickly harvested and fixed in situ via the trachea cannula with buffered4%formaldehyde, and then were embedded in paraffin. The sections were cut into4-5μm slices and were stained with streptavidin peroxidase and hematoxylin-eosin (HE). The structural changes in pulmonary vascular wall were observed by immunofluorescence microscopy and wall thickness (MT), blood vessel diameter (ED), pulmonary artery lumen area (VA), and vascular area (TAA) were measured to calculate MT%(=MT/ED), VA%(=VA/TAA). The ratio of right ventricular to body weight (RV/BW) was determined to measure the right ventricular hypertrophy. According to the results of hemodynamic parameters, right ventricular hypertrophy, pulmonary arterial pathological changes to evaluate if the model of pulmonary arterial hypertension was successful establishment. 2. Isolation, culture, immunophenotype analysis and labeling of MSCs2.1Isolated and cultured of bone marrow cellsBone marrow cells were isolated by flushing the cavity of femurs and tibias and transferred to a tissue culture dish90mm in diameter. To separate BMSCs and other cells, the Ficoll (1.077) density gradient centrifugation method was used. The white coat composed of mononuclear cells from the upper layer and interface was carefully collected and washed three times. The isolated bone marrow cells were then cultured and expanded in low glucose culture containing Dulbecco's modified Eagle medium/F-12, supplemented with10%fetal bovine serum,100U/mL penicillin and100g/mL streptomycin. When adherent cells were confluent, they were continuously cultured until passage3to5using trypsin (0.25%)/1mmol/L ethylenediamine-tetraacetic acid (EDTA) for5minutes. All culture experiments in this study were maintained at37℃and in5%humidified CO2and the cultures were replenished with fresh medium every3days.2.2BMSCs immunophenotype analysisFlow cytometric immunophenotyping was performed by using methods reported previously:5×105cells were suspended with trypsin and were washed2times in phosphate-buffered saline (PBS). After centrifugation, the cells were incubated with primary against human CD29, CD90, CD34, CD45antibody for30min at4℃. The second polyclonal antibody-FITC conjugated against mouse IgG was added and incubated at4℃for an additional30min in a dark room. The same isotype irrelevant antibody of the same species was used as negative control. After being washed, cells were resuspended in PBS for FACS (Fluorescence-activated cell sorter) analysis.2.3BMSCs label in vitroBefore implantation, the cells were labeled with the cross-linkable membrane dye CM-DiI (1μg/mL) according to the protocol of the supplier as previously described. Briefly,1-5×105cells were incubated for5min at37℃, and then for an additional15min at4℃. After labeling,1×105cells were being washed with PBS and resuspended in100μL saline and then kept on ice before transplantation, the labeling-efficiency reached more than80%. 3. Groups of experimental animalsThe male SD rats (n=30) were randomly assigned to3groups (n=10in each group) as follows:Control group:animals that received a abdominal injection of0.9%saline instead of MCT;PAH group:animals that received a abdominal injection of60mg/kg MCT;BMSCs group:animals that received1-5×105labeld BMSCs implantation one week after MCT injection.4. Effect of BMSCs on PAH model4.1BMSCs transplantationOne week after MCT injection, the rats were anesthetized with subcutaneous injection of pentobarbital,1-5×105CM-DiI labeld BMSCs were resuspended in100μL saline and were administration by sublingual vein.4.2Immunological and immunohistochemical analysisThe rats were euthanized and the left lung inflated with OCT compound, and then quickly frozen in liquid nitrogen and stored at-80℃. Sections were cut into4μm slices and fixed in acetone for10min at-20℃. Survival of BMSCs was demonstrated by observing the presence of DiI-labeled cells. Immunofluorescence was then carried out with goat anti-mouse monoclonal surfactant associated protein C (SP-C,1:100) IgG antibody, rabbit anti-human von Willebrand Factor (vWF,1:100) antibody and vascular endothelial growth factor (VEGF,1:100) anti-body. FITC-conjugated antiserum (1:200) was used as a secondary antibody. Some of the tissues were fixed with4%formaldehyde and for paraffin embedding.Three weeks after BMSCs transplantation the rats were anesthetized and lung inflated with OCT compound, and then quickly frozen in liquid nitrogen and stored at-80℃. Sections were cut into4μm slices and fixed in acetone for10min at-20℃Survival of BMSCs was demonstrated by observing the presence of Dil-labeled cells. Immunofluorescence was then carried out with goat anti-mouse monoclonal surfactant associated protein C (SP-C,1:100) IgG antibody, rabbit anti-human von Willebrand Factor (vWF,1:100) antibody and vascular endothelial growth factor (VEGF,1:100) anti-body. FITC-conjugated antiserum was used as a secondary antibody.4.3Assessment of hemodynamics and effect of BMSCs on RV Impairment Induced by MCTThree weeks after BMSCs transplantation, the rats were anesthetized and inserted with a3F-Miller microtip catheter via the right jugular vein into the right ventricle to obtain base line measurements of hemodynamics such as right ventricular systolic pressure (RVSP), mean right ventricular pressure (MRVP), and mean pulmonary arterial pressure (MPAP). The ratio of right ventricular to body weight (RV/BW) was determined to measure the right ventricular hypertrophy.4.4The structural changes in pulmonary vascular wallThe lung was quickly harvested and fixed in situ via the trachea cannula with buffered4%formaldehyde, and then was embedded in paraffin. The sections were cut into5μm slices and were stained with streptavidin peroxidase and hematoxylin-eosin (HE). The morphometric analysis of pulmonary arteries as described previously.Results:1. Evaluation of animal survivalOne week after abdominal injection of monocrotaline in the rats, eating and activity reduction, unresponsive, hair is lacklustre, shortness of breath, weight loss, weight loss or gain. While three weeks after BMSCs transplantation the characteristic is not obvious, the general condition was good.2. The analysis of MCT-Induced PAH model2.1Morphological observationOne week after abdominal injection of monocrotaline in the rats, gross observation demonstrated that the lungs enlarged volume, colour purple, the surface is uneven, poor elasticity, and partial visible blood stasis in PAH group compared with normal control group.2.2Hemodynamics and RV ImpairmentOne week after MCT injection, RVSP, MRVP and MPAP were significantly elevated in PAH group compared with control (P＜0.05), the ratio of RV/BW was significantly higher in the PAH group compared with control group (0.49±0.03 vs.0.57±0.06, P<0.05). These results indicated that MCT could lead to severe right ventricular hypertrophy and the PAH model was successful established.2.3Pulmonary artery wall observationOne week after MCT injection, hematoxylin and eosin (H&E) staining demonstrated a significantly intima thickening and luminal stenosis in PAH group compared with Control group. MT%muscular arteries was significantly increased and VA%was significantly decreased in PAH grpup as compared with control group, respectively (38.27±4.55vs.13.05±1.33and33.39±6.63vs.44.09±4.17, P＜0.01).3. Effect of BMSCs on MCT-induced PAH3.1Hemodynamics and RV ImpairmentThree weeks after BMSCs administration, RVSP, MRVP and MPAP were significantly lower in BMSCs group compared with PAH group (P＜0.05), the ratio of RV/BW was significantly lower BMSCs group compared with PAH group.3.2Pulmonary artery wallHematoxylin and eosin (H&E) staining demonstrated that the intima thickening and luminal stenosis were significantly decreased in BMSCs group compared with PAH group. MT%of muscular arteries was significantly reduced in BMSCs group as compared with PAH group (P<0.05),and VA%of muscular arteries was significantly improved in BMSCs group as compared with PAH group (P<0.05)。4. Identification of the transplanted MSCsThree weeks after BMSCs implantation, DiI-labeled red fluorescence-positive cells were observed at the transplanted area in BMSCs group. In many regions, the red fluorescence-positive cells were observed coincident with the green fluorescence spots of VEGF and vWF anti-body but not SP-C anti-body, which suggested that the intravenous implantation of BMSCs could differentiate into vascular endothelial cells in vivo although they did not actively survive as lung cells. There was no evidence of red and green fluorescence in control and PAH groups.Conclusion:1. Combined application of density gradient centrifugation method and adherent culture method acquired enough highly purified activated BMSCs.2. The transplanted BMSCs still alive after3weeks, and part of the experiment appears to have differentiated into pulmonary vascular endothelial cells. 3. Intravenous implantation of BMSCs can significantly reduce or even reverse the progression of MCT-induced pulmonary arterial hypertension, improvement cardiac function and hemodynamics.