Autologous Adipose Derived Stromal Cells Transplantation Ameliorate Hyperkinetic Pulmonary Arterial Hypertension

Background:Hyperkinetic pulmonary arterial hypertension (PAH) is the common complication of congenital heart disease (CHD) with left to right shunt. The opportunity of surgery, success of surgery and the quality of postoperative life were seriously effected by PAH. Now, studies on the mechanism and therapy of PAH have made great progress. It has been recognized that PAH was characterized by an increased resistance in pulmonary circulation and the occlusive remodeling of the pulmonary arterioles. Pathologically, hypertrophy of the media in pulmonary arterioles contributed to the occlusion of the vessels and ultimately, made the decrease of pulmonary vascular bed. Even worse, the increased pulmonary arterial pressure developed higher than before and then deteriorated the pulmonary function. In some cases, pulmonary hypertension induced by CHD could be reversed after surgical treatment. Vasodilation therapy has improved the life quality of patients with PAH. While the obstructive remodeling occurred, mere vasodilation treatment or the surgery could not reverse the pathological changes in the lung. Recent years, therapeutic angiogenesis and the tissue engineering cells for vascular regeneration have been a hot spot in the medical field. Adipose derived stromal cells (ADSCs) as a multipotential stem cell attracted the interests of researchers for its advantages such as the minor invasion and easy culture. ADSCs could secrete some cytokines such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) for angiogenesis. HGF was an efficient vasculogenesis factor and could restore the injured organ. Many studies about the application of ADSCs in the ischemia diseases have made success. Previous study has proved that ADSCs implantation could improve the damaged lung in animal models such as emophysematous models via participating in the regeneration of vessels and alveolus or through secreting some cytokines. The perfusion of lung was improved and subsequently the gas exchange function of lung was also restored. But, whether the transplantation of ADSCs could restore the lung of the PAH models has not been reported. So, we put forward the hypothesis that the autologous ADSCs transplantation might improve the function of lung under PAH situation.Objective:To establish a hyperkinetic PAH model, to isolate the ADSCs from rat fat, to label the ADSCs in vitro efficiently before injected, to investigate the hemodynamics and the pathological changes of pulmonary arterioles and to analyze the expression of HGF in the lung for explaining the possible reason for the changes of pulmonary arterial pressure after ADSCs tansplantation.Methods:1. Carotid artery-jugular vein shunt was performed using a cannulation style in rat. Echocardiogram was performed to measure the internal diameter of pulmonary valve and aortic valve, frequency spectrum of pulmonary flow and the thickness of right ventricular anterior wall (RVAW) and left ventricular posterior wall (LVPW) and to investigate right ventricle by apical four chamber view. Invasive examination was the right heart catheter and thoracotomy for the measurement of pulmonary arterial pressure. Pathomorphyology analysis of pulmonary arterioles included vascular wall thickness index (TI), relative vascular wall area index (AI), and the percentage of the muscularized vessels. 2. The fat was obtained form rat inguina and epididymis. A series of filtration and centrifugation was performed after the digestion of collagenase for the isolation of adipose derived stromal cells (ADSCs). Morphous, the immunophenotype, and the multi-directional differentiation of ADSCs such as adipogenic, osteogenic inductions were investigated. Growth curve of ADSCs was draw after MTT test and the doubling generation time was calculated. Enzyme linked immunosorbent assay (ELISA) was used for detecting the HGF in supernatant of ADSCs cultured under hypoxia or normaxia condition in vitro. Cells were labeled by CM-DiL and MTT test was also performed to investigate the cell vitality after labeling.3. The data of the PAH models 12 weeks after A-V shunt was regarded as the baseline data. Another PAH models were separated into blank group (without any treatment after shunt surgery), cell transplantation group (5×107 cells in 0.5 mL DMEM were injected per rat through right jugular vein), DMEM group (injection of 0.5 mL DMEM) and the sham group was taken as negative control. Two weeks after cell transplantation, Echocardiogram, right heart catheter and gas blood ananlysis were performed. Frozen slices and immunofluorescence was made for investigating the location of the transplanted cells and HGF secreted by ADSCs in vivo. Immunohistocytochemistry of HGF and VIII factor were also performed to compare the vascular number among the groups for revealing the possible mechanisms of the decreasing of pulmonary arterial pressure. Real time PCR and Western blot analysis were done to demonstrate the expressions of mRNA and protein levels of HGF and eNOS.Results:1.Ten rats died because of the overdose of anesthesia drugs or bleeding during the operation. The surgery mortality rate was 13.4%(10/60). Four rats died of the loss of cannulation or other accidents. The postoperative mortality rate was 8% (4/50). In the 46 residual living rats,12 animals were excluded from the study because occlusion of the shunt which was detected by echocardiogram. The patency rate was 73.9% (34/46). Twelve weeks after the shunt surgery, echocardiogram showed that the internal diameter of pulmonary valve (3.52±0.27mm) was much wider than the internal diameter of aortic valve (2.99±0.32mm, P<0.05) in animals underwent shunt operation. Pulmonary arterial blood frequency spectrum analysis suggested that the pulmonary arterial acceleration time (PAAT) of the shunt rats (75.31±22.12 ms) was much shorter than that in the normal rats (135.14±26.42 ms) and that in the 4th week (119.38±12.81 ms) and 8th week (106.56±31.45 ms) rats after operation. Twelve weeks after shunt operation, the rate of RVAW to LVPW increased (63.02±14.36%) compared with that in the normal control animals (41.08±4.54%) or that in the 4th week (40.46±13.41%) and 8th week (42.24±5.87%) animals. The enlargement of right ventricle was shown by apical four chamber view in the animals after 12 weeks shunt and the interventricular septum shifting toward left side during systolic period was also displayed by echocardiogram. Pulmonary arterial pressure was higher in the shunt rats (37.69±7.81 mmHg)than that in the normal animals (16.59±4.51mmHg, P<0.05) which was confirmed by the right heart catheter and the thoracotomy for the pressure measurement. Pathological examination for the pulmonary arterioles showed that the TI and AI were both increased in the shunt animals (34.25±9.11% and 69.72±13.19%, respectively) than that in the normal rats (16.71±4.99% and 29.05±9.79%, respectively, P<0.05)2. ADSCs from the rat's adipose tissue exhibited a fibroblast-like morphology, had the ability to self-renew and adhere to plastic 72 hours after the planting, and extensively expanded in culture without loss of differentiation potential. These cells could be induced into mature adipocytes, which was confirmed by microscopic observation of intracellular lipid droplets after Oil Red O staining. ADSCs also differentiated into osteoblasts, which was evaluated by alkaline phosphatase staining. Immunocytochemistry revealed that these cells showed a positive signal for factor-Ⅷin vitro after the endothelial induction. Fluorescence-activated cell sorter analysis showed that the cultured ADSCs were positive for stem cell such as CD29, CD105 and SCa-1. The percentage of CD31 positive cells was a little higher in the primary cells and decreased after twice passages. MTT examination showed that the ADSCs have high vitality and the doubling generation time was between 24-60 hours since the planting. During the passage, trypan blue stain showed that the rate of living cells maintained over 90% within the passage 15. ELISA results demonstrated that the HGF content in supernatant of the cells cultured under hypoxia condition was much higher than that of the cells cultured under normoxia condition. Furthermore, the younger the cell is, the more the HGF was detected in the supernatant. The labeling rate of CM-DiL in ADSCs was more than 99% and the fluorescence did not quench even after several passages. In addition, the MTT test for the labeled cells with CM-DiL showed that no difference in the growth curve and the doubling generation time compared with the normal cells.3. Two weeks after the ADSCs transplantation, the PAAT was extended than that in the blank control group (129.58±35.14 milliseconds VS 80.49±21.29milliseconds, P<0.05). The decreased size of right ventricle and the disappearance of interventricular septum shift were displayed in the apical four chamber view by echocardiogram and the rate of RVAW/LVPW was also significantly lower compared with that in the blank control group (42.63±8.71% VS 59.39±7.12%, P<0.05) Pulmonary arterial pressure in cell transplantation group (19.83±2.32mmHg) was decreased than that in the blank control group (35.82±5.09 mmHg, P<0.05). The transplanted ADSCs located around the vessels in lung which was observed under fluorescence microscope. Immunofluorescence for the HGF analysis indicated that the transplanted cells secreted HGF in vivo. Furthermore, the cells transplantation augments the HGF expression in lung which was confirmed by RT-PCR and Western blot analysis. In addition, the expression of eNOS in lung was also increased after cell transplantation which was in accordance with the increasing of the HGF. Finally, VIII factor immunohistochemistry stain showed that the vessels in certain area of the lung were much more than that in the blank control group.Conclusion:1. Carotid artery-Jugular vein shunt in rat could establish a hyperkinetic pulmonary arterial hypertension model after 12 weeks if the patent was patent. The pathological alteration conforms to the pre-tricuspid valve hyperkinetic pulmonary hypertension.2. ADSCs could be obtained from rat fat. ADSCs were in conformity with the standard of stem cells on the appearance, immunophenotype and the multidirectional differentiation. ADSCs were of high vitality even in high passage cells. ADSCs cultured under hypoxia condition secreted more HGF than that in cells cultured under normoxia condition. CM-DiL was a good tracking probe for the cell transplantation.3. Autologous ADSCs transplantation ameliorated pulmonary hypertension induced by shunt flow, reversed the deteriorative hemodynamics caused by PAH, and decreased the pathological process of the pulmonary arterioles, therefore, improved the gas exchange function of lung. All the results mentioned above might due to the HGF secretion of ADSCs and subsequently the increased HGF improved the angiogenesis and the eNOS expression in the lung tissue.