Therapeutic Effects Of Mesenchymal Stem Cells Transplantation On Pulmonary Arterial Hypertension And Influence On Expression Of Endothelin-1 In Rat Models

Pulmonary artery hypertension(PAH) is a common cadiovascular disease characterized by increased pulmonary arterial pressure and pulmonary vascular resistance, patients with it ultimately died of right heart dysfunction. It's difficult to treat and has a slow development due mainly to complicated etiological factors. Although many therapies, including intravenous PGI2, endothelin receptor antagonist and NO inhalation have proven useful in decreasing pulmonary arterial pressure, improving exercise tolerance and quality of life, the long-term outcome in this disorder is bleak.Now regeneration and gene therapy are used to interrupt vicious cycle of PAH.To reproduction healthy endothelial cells, bone marrow cells such as endothelial progenitor cells, endothelial progenitor like cells and mesenchymal stem cells are grafted into vivo.It's also being attempted to transfect adrenomedulin or eNOS to improve efficacy by adding pulmonary beds and promote endothelial cells'secretions (NO).Recently it's a heat spot of stem cells transplantation to treat PAH.The issues are still not clear such as what's the safe way to graft,how many cells are safe and have optimal effects on PAH, and how the endothelin-1 (ET-1) changes after cells transplantation.Endothelin-1, a 21-amino acid polypeptide produced by vascular endothelial cells, has powerful effects of cell differentiation and proliferation and the most powerful vasoactive properties hitherto.ET-1 cannot regulate normal pulmonary tone,however,it predominance at diseases.It can develop PAH directly by contracting pulmonary vessels, stimulating vascular smooth muscle cells and fibroblast to proliferate, promoting platelet aggregation and by producting hyperoxide. Therefore, plasma and tissue ET-1 is both one of the mechanisms of formation and a sign of seriousness in pulmonary hypertension.In this study, we aim to establish rat's mesenchymal stem cells system and pulmonary hypertension model induced by monocrotaline,and to study what impacts that different numbers of MSCs are transplanted intravenously on pulmonary hypertension induced by MCT. We examinine RVSP, RV/LV+S weight ratio and pathological changes of small pulmonary arteries in lung tissue.Also the prepro-ET-1 mRNA in lung and ET-1 in serum are tested.we can determine the impact of MSCs on the PAH and find safe, appropriate number of transplanted cells,and provide a new method of treatment on PAH and it's theoretical basis. Cultivation,identification and labelling of mesenchymal stem cells in Wistar ratsMethods:MSCs of Wistar rats were isolated and cultivated by Bone marrow adherent culture.The morphology characters of MSCs were observed under phase contrast microscope. The expression of CD29, CD44, CD34 and CD45 of the cells were analyzed by flow cytometry. Ultrastructures were observed by electron microscopy. Osteogenic differentiation was studied by alkaline phosphatase (AP) staining.MSCs were labeled by DAPI and were traced for cell transplantation in vivo.Results:After 24 hours' primary culture, MSCs adhered to plastic surface of the culture dish,and proliferated in colonies in 6-8 days.These primary MSCs reached 80%～90% of confluence in 12～15 days.The positive rates of CD29,CD44, CD34 and CD45 in MSCs at P3 (third generation) were 98.6%,78.2%,0.0% and 0.3% respectively.Parts of cultured expanded MSCs at P3 cultivated in Osteoblast induction medium showed positive by alkaline phosphatase staining in 14 days.MSCs' nulears can be labeled by DAPI expressing blue fluorescence.Pathological changes of pulmonary arterial hypertension by two different doses of monocrotaline in ratsMethods:Male Wistar rats(n=70) were randomly devided into three group:control group(n=10), 50mg·kg-1 MCT group(n=30) and 60mg·kg-1 MCT group (n=30).At two weeks and four weeks after injected intraperitoneally with MCT(injected intraperitoneally with equal normal saline),right ventricular systolic pressure(RVSP)and right ventricle weight/(left ventricle+septum) weight [RV/(LV+S)] ratio were measured.Lung tissues were stained by hematoxylin-eosin staining and orcein technique to observe the pathological changes of lung and medial thickness percents of pulmonary arterioles.Ten rats in each MCT group were recorded the number of days of natural death.Results:Regardless of two weeks or four weeks after MCT administration,RVSP in 50mg·kg-1 group were higher than that in control group (respectively 36.6±5.1mmHg, 39.1±7.0mmHg versus 26.1±3.8mmHg, both P≤0.01),and RV/(LV+S) weight ratio increased greatly (respectively 0.2894±0.0217,0.4094±0.1256 versus 0.2473±0.0193,both P<0.01),so were the results in 60mg-kg-1group contrast to the control group:RVSP (respectively 36.8±5.5mmHg,46.6±12.6mmHg versus 26.1±3.8mmHg, both P＜0.01); RV/(LV+S) weight ratio:(respectively 0.3082±0.0584,0.4175±0.1037 versus 0.2473±0.0193,both P±0.01).Yet no significant differences were observed of RVSP and RV/LV+S weight ratio between the two different doses of MCT group. The pulmonary arterioles were markedly thickened in both 50mg-kg-1 and 60mg-kg-1 PAH rats contrast to in control ones[(21.3±1.9)%,(23.4±2.3)% versus (11.3±1.2)%,respectively P＜0.01].Rats induced by 60mg-kg-1 MCT were half dead by day 47 and none survived by day 63,yet none of rats induced by 50mg-kg-1 MCT was dead by day 47 and seventy percent survived by the day 63.Therapeutic effects of mesenchymal stem cells transplantation for pulmonary arterial hypertension and influence on the expression of endothelin-1 in rat modelsMethods:we cultivated wistar rats,7 days old,and prepared the third generation cells for cell transplantation.68 free male wistar rats (180-250g) were used for experiment.(1) Twenty rats divided into four groups according to the numbers of transplantation cells through external jugular vein:the 5×105 group,the 1×106group,5×106group and the control group (physiological saline) and each group were five.RVSP were measured before transplantation, 5 min,30min,and 24 hours after transplantataion respectively;(2) Eight rats were divived into two groups by cells transplantation ways,one was intravenous administration (n=4) and another was intratracheal administration (n=4).1×106 MSCs labeled by DAPI were transplanted into rats induced by 60mg-kg-1 MCT.Three weeks later,lung tissues were harvested for frozen section to observe the labeled cells.(3) Forty rats were divived randomly into four groups(each group n=10):①the MCT/MSCs 5×105 group;②the MCT/MSCs 1×106group;③the MCT group;④the control group.The rats were injected intraperitoneally with 60mg-kg-1 MCT,while the control group with physiological saline.And 1 mL PBS Containing 5x105MSCs were transplanted into rats through external jugular vein in first group,1×106MSCs in the second and 1 ml PBS in the last two groups respectively.and On the four weeks after cell transplantation,the RVSP and RV/(LV+S) weight ratio were measured;Lungs'tissues were stained by Hematoxylin-eosin staining, lichen dyed red staining and smooth muscle Actin Immunohistochemistry, and lungs'Pathological changes and the thickness of pulmonary arteries percentage were observed.The expression of endothelin-1 was determinated by immune ELISA in the serum separated from the abdominal aorta. Also the expression of the prepro-ET-1 mRNA was observed by RT-PCR in the lungs. The statistics data were analyzed by One-way analysis of variance using SPSS 16.0.Results:(1) Intravenous administration of 5x105 MSCs and physiological saline was safe and Almost could not significantly increase RVSP in rats.However.RVSP rised from 22.3±4.4 mmHg prior to intravenous implantation of 1×106 MSCs to 35.8×5.6 mmHg 5 min after it,to 44.2±4.5mmHg 30 min after,and only delined to 27.8±3.6 mmHg 24 hours after administration,which was slightly higher than control rats.It was unsafe to administration of 5×106 MSCs,because of high mortality;(2) There were plenty of MSCs with blue fluorescence in lungs 3 weeks after MSCs which labeled by DAPI were implanted intravenously,However there were high mortality rate and very little blue MSCs in Survival rats with intratracheal transplantation.(3) At 4 wk,RVSP was 47.2±10.5 mmHg in MCT/MSCs 1×106 rats and 35.8±8.4 mmHg in MCT rats,there was Significant difference(P<0.05),and ventricular weight ratio was 0.3572±0.0923 also significantly lower than 0.4454±0.0935 in MCT rats(P<0.05). However,in MCT/MSCs 5×105 rats,RVSP was 42.5±11.3mmHg and ventricular weight ratio was 0.4003±0.0725,both not Significantly decreased compared to those in MCT rats (both P>0.05).(4) By pathological staining, the percentage of media thickness of the pulmonary arterioles were observed significantly thinner in MCT/MSCs 1×106 group than those in MCT group.(19.2±3.8%, vs.26.4±4.9%, P<0.05),but it was not thinner in MCT/MSCs 5±105 group than MCT group (23.3±3.6%, vs.26.4±4.9%,P>0.05).(5) RT-PCR detection showed that prepro-ET-1 mRNA expression was little in control rats while was high in MCT rats.And MSCs' administration could decrease its expression, more significantly in MCT/MSCs 1×106 group than in MCT/MSCs 5×105one(P<0.05). The results were similar through analyzing expression of endothelin-1 in the serum by ELISA.This research found that when mesenchymal stem cells were transplanted to rat model in PAH induced by MCT,it was safe to transplant 1×106 or 5×105 MSCs through the external jugular vein way, but not safe that 5×106 MSCs were transplanted intravenously.It was also not suitable transplant MSCs by intratracheal administration.MSCs could improve rats' RVSP and RV/LV+S in PAH induced by MCT. And also they could reduce inflammation,medial hypertrophy in lungs'arteries. The expression mRNA of prepro ET-1 in lung tissues and plasma ET-1 protein were decreased.So we concluded 1×106 MSCs had significantly more effective role than 5×105 ones on preventing PAH.In this study we identified and labeled MSCs by means of observing cell morphology, electron microscopy, flow cytometry, fluorescence microscopy.we also identified rats PAH model with measuring RVSP,pathological HE staining and Lichens red staining.The effects of MSCs'administration on PAH induced by MCT were studied by pathological staining, immunohistochemistry,ELISA, RT-PCR and Western-blotting.The pathological changes had improved and the expression of prepro ET-1 mRNA and ET-1 protein in plasma had decreased significantly after MSCs administration. Because ET-1 was an important molecule, a mark of severity and a mechanism for development in PAH, we could evaluate the effects of MSCs administration on preventing PAH by molecular level.