Effects Of Pioglitazone And Insulin On Endothelial Progenitor Cells Derived From Bone Marrow Of Rat

Background and aimsRecently, more and more researches of stem cells including endothelial progenitor cells (EPCs) are turning to the treatment of coronary heart disease (CHD) and diabetes (DM). EPCs are bone marrow-derived progenitor cells that express surface markers such as AC133,CD34 and VEGFR-2(flk-1), and can differentiate into mature endothelial cells on the vascular wall by the way of secreting VEGF or homing to injured areas. So, EPCs play an important role in repair of endothelial injuries and revascularization of infarcted areas. Many researches indicate that the number of circulatory EPCs is the index of predicting cardiovascular functions and risk factor of cardiovascular diseases.Smaller number means lower capacity of repairing endothelia and higher morbidity of cardiovascular diseases. The number of EPCs in patients suffering from CHD and DM decreases, so does the capacity of proliferation, migration, homing and angiogenesis of EPCs. As a result, increasing the number and function of EPCs with drugs in order to promote the process of repairing endothelia and revascularization becomes a new method of treating cardiovascular diseases. One of these drugs mentioned above is insulin. Insulin can activate endothelial nitric oxide synthase (eNOS), which secretes nitric oxide (NO) and dilates blood vessels. Recent researches have showed that eNOS is an indispensable factor in mobilization and modulation of stem cells. Another drug is pioglitazone, which is a kind of thiazolidinediones (TZDs) and shows good effects on decreasing blood glucose and excellent tolerance. A recent study illustrates that pioglitazone could promote angiogenesis of EPCs, but the mechanism is still unknown.This study intends to explore the effects of pioglitazone given in vivo with different dosages and insulin given in vitro on the number, proliferation, apoptosis and secretion of NO of EPCs derived from bone marrow, and the results may help to understand the cardiovascular effects of pioglitazone and insulin from a new aspect.MethodsPartⅠ: Culture and identification of surface markers of EPCs. 1. Harvest bone marrow of rats, and collect mononuclear cells through density gradient centrifugation. Then culture them in Medium 199 supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). 2. Immunofluorescent co-stain with DiI-ac-LDL and FITC-UEA-I to identify EPCs.PartⅡ: Effects of pioglitazone with different dosages given in vivo on EPCs. 1. Sprague-Dawlay (SD) rats are divided into four groups randomly. The control group was given saline by intragastric administration, and the three other groups are given pioglitazone by 10, 20, 40 mg·kg-1·d-1 respectively for 10 days. 2. Mononuclear cells were collected from rats'bone marrow by density gradient centrifugation. 3. Once being harvested, EPCs of each group were calculated and cultured with Medium 199. 4. Test the proliferation ability of EPCs by MTT assay. 5. Detect the apoptosis level of EPCs by immunofluorescent co-staining. 6. Measure the secretion of NO by modified Griess reaction method after 24 h of incubation.PartⅢ: Effects of pioglitazone and insulin on EPCs. 1. SD rats are divided into two groups randomly. The non-pioglitazone group is given saline by intragastric administration, and the pioglitazone group is given pioglitazone (20 mg·kg-1·d-1 ) for 10 days. 2. Mononuclear cells were collected from rats'bone marrow by density gradient centrifugation. 3. Once being harvested, EPCs of each group were divided into two subgroups, with one subgroup being given solvent and the other insulin (1nmol/L). All EPCs of these four groups were calculated and cultured with Medium 199. 4. Test the proliferation ability of EPCs by MTT assay. 5. Detect the apoptosis level of EPCs by immunofluorescent co-staining. 6. Measure the secretion of NO by modified Griess reaction method after 24 h of incubation.Statistical analysis is performed with SPSS version 13.0. One-way analysis of variation and post hoc t (LSD-t) test are employed.Results1. After being cultured for 7 days, cells obtained showed double positive for DiI-ac-LDL and FITC-UEA-I, which indicated that they were EPCs.2. Pioglitazone of different dosages increased the number and function of EPCs.3. Pioglitazone of 20 mg·kg-1·d-1 showed better effects of promoting proliferation and secreting NO of EPCs than the groups of 10 mg·kg-1·d-1 and 40 mg·kg-1·d-1 but same effects of inhibiting apoptosis as these two groups.4. Both pioglitazone and insulin had effects of promoting proliferation and secreting NO and inhibiting apoptosis of EPCs. 5. Insulin showed better effect than pioglitazone in promoting proliferation but same effects of inhibiting apoptosis and promoting secretion of NO of EPCs as pioglitazone.6. Co-application of pioglitazone and insulin showed better effects of promoting proliferation and secreting NO and inhibiting apoptosis of EPCs than single use of each drug.Conclusion1. Pioglitazone of different dosages can increase the number, promote proliferation and secreting NO and inhibit apoptosis of EPCs.2. Both pioglitazone of 20 mg·kg-1·d-1 and insulin have effects of promoting proliferation and secretion of NO and inhibiting apoptosis of EPCs, and they have synergistic effects in these aspects.