Effects And Mechanisms Of Endothelial Progenitor Cells On Pulmonary Production Of Prostacydin By Paracine Action

Background and AimsIdiopathic pulmonary artery hypertension (IPAH) is an idiopathic malignant pulmonary vascular disease which characterized by a particularly severe arteriopathy, including intimal hyperplasia, muscularization of the distal alveolar duct and pulmonary arteries, plexiform lesion and neointima which obstruct the pulmonary arteries and arterioles, eventually leading to severe PAH, right heart failure.Vascular endothelial cells and smooth muscle cells play an important role in the development of pulmonary hypertension. It is considered that the change of structure, function and metabolism in endothelial cells is an important feature in the onset and progression of PAH. The damage and activation of endothelial cells and the synthesis of a variety of factors are involved in the vasoconstriction and reconstruction of pulmonary artery. The vasodilator substances released by the injured endothelial cells, such as nitric oxide (NO), prostacyclin (PGI2) are reduced; however, the vasoconstrictor substances such as endothelin-1(ET-1), angiotensin (Ang) are increased. Therefore, the balance between endothelium-dependent vasodilators and vasoconstrictors is the pathophysiological basis of PAH.Endothelial progenitor cells (EPC) are a precursor cell population which could differentiate into mature endothelial cells, but do not acquire mature endothelial lineage markers or form a lumen yet. EPC have the capacity to be involved not only in human embryonic angiogenesis, but also involved in the process of postna tal angiogenesis and endothelial repair. In recent years, transplantation of EPC in the treatment of PAH attracts great interest and has become a new hot spot. Animal experiments have shown that in monocrotaline (MCT)-induced PAH rats and dogs, given EPC almost completely prevented the increase in right heart systolic pressure (RVSP). Furthermore, EPC have been used to treat clinical IP AH patients in our pilot study showing some short-term efficacy. Although there is increasing experimental evidence for the involvement of EPC in neovascularization and vascular repair, the exact underlying mechanisms are still poorly understood. Previous studies mainly reported that the transplanted EPC were directly involved in angiogenesis and endothelial repair. However, there is no lack of angiogenesis in patients with PAH and some patients even have endothelial hyperplasia (plexiform lesions). Therefore, angiogenesis may not be sufficient to fully account for the protective effect of EPC, suggesting that paracrine mechanisms may play an important role. Thus, the purpose of our study was to investigate whether bone marrow-derived endothelial progenitor cells (BMEPC) could stimulate the production of endothelium-dependent vasodilators in pulmonary artery by paracrine action and further to explore its mechanisms.MethodsMononuclear cells were obtained from bone marrow of normal Fisher-344rats and cultured for7days to yield BMEPC. ELIS A was used to test the level of vascular endothelial growth factor (VEGF) in BMEPC-Conditioned medium (BMEPC-CM).In vitro study,50adult F344rats were divided randomly into two groups:control group(n=10) and MCT group(n=40). To induce PAH, rats were injected a single intraperitoneally of MCT (60mg/kg body weight, BW). Control animals were injected intraperitoneally with saline. The degree of PAH was assessed21days after MCT or saline injection by measuring RVSP. After24hours exposure to BMEPC (5×106cells), pulmonary arteries were isolated for examining vascular reactivity. The proteins expression of cyclooxygenase-2(COX-2), cyclooxygenase-1(COX-1) and prostacyclin synthase (PGIS) were determined by western blot. The content of adenosine cyclophosphate (cAMP) was tested by radioimmunoassay (RIA).In vivo study,21days after MCT injection, rats were transplanted once with BMEPC (5×106)(BMEPC group, n=15), BMEPC-Conditioned medium (BMEPC-CM group, n=15). The control groups were not given MCT nor cells (Control group, n=15) or were given MCT injection only (MCT group, n=15). Another21days later, the body weight, RVSP, Lung/Body Weight (L/BW), Right ventricular/left ventricular and septum weight (RV/LV+IVS) and morphometric changes were examined respectively. Western blot analysis were employed to determine the proteins expression of COX-2, COX-1, PGIS, endothelial NO synthase (eNOS), inducible NO synthase (iNOS). The content of6-keto-Prostaglandin F1α (6-keto-PGF1α), Prostaglandin E2(PGE2), Thromboxane B2(TXB2) and cAMP were examined respectively by enzyme-linked immunosorbent assay (ELISA) and RIA. Finally, immunohistochemical analysis was used to observe the expression and distribution of COX-2.ResultsIn vitro experiment, on the21st day after MCT injection, rats became weak, lethargic and showed a dull coat. The level of vascular endothelial growth factor (VEGF) was significantly higher in BMEPC-CM compared with bone marrow derived mononuclear cells-conditioned medium (BMMNC-CM). After exposure to BMEPC, acetylcholine (ACH)-induced relaxation in pulmonary arteries of MCT-induced PAH was improved, and the change was inhibited by COX-2inhibitor, NS398. However, BMMNC did not have this effect. Western blot showed that the proteins expression of COX-2and PGIS were increased in pulmonary arteries treated with BMEPC. And the content of cAMP was significantly enhanced.In vivo study, on the42nd after MCT treatment, body weight was lower in MCT group than control group (265.17±4.48g vs.329.33±15.18g, P＜0.01). This decrease was not significantly prevented by BMEPC treatment. MCT-treated rats had a significantly increased RVSP on day42(62.37±1.98mmHg) in comparison with control rats (25.42±0.95mmHg). However, the delivery of BMEPC prevented the increase in RVSP. Moreover, BMEPC transplantation attenuated the increase in L/BW and RV/LV+IVS ratio compared to MCT group (0.59±0.03vs.0.41±0.04, P＜0.01). Histological examination revealed that, MCT induced vascular remodeling characterized by vascular medial wall thickening in both small and moderately sized pulmonary arteries, resulting in lumen narrowing or near obstruction. However, BMEPC treatment markedly attenuated this change. Further analysis demonstrated that the proteins expression of COX-2and PGIS were increased in pulmonary arteries treated with BMEPC. The release of6-keto-PGF1α and cAMP were significantly enhanced, and the change was only reversed by a selective COX-2inhibitor, NS-398. Moreover, the increased protein expression of COX-2was observed in all three layers of pulmonary arteries transplanted with BMEPC.ConclusionsBMEPC could improve the injured ACH-induced relaxation in pulmonary arteries of MCT treated rats. Transplantation of BMEPC could effectively ameliorate MCT-induced PAH and improve the remolded pulmonary arteries. Growth factors like VEGF, secreted by BMEPC may promote vasoprotection in a paracrine manner by increasing the release of PGI2and content of cAMP.