RESUMO
<p><b>OBJECTIVE</b>To explore the proangiogenic activity of exsomes released by human umbilical cord mesenchymal stem cells (MSCs) stimulated by erythropoietin and platelet-derived growth factor BB (PDGF-BB).</p><p><b>METHODS</b>Human umbilical cord-derived MSCs were seeded and maintained in culture overnight. The media were then replaced by alpha-MEM containing EPO (1 U/ml) and/or PDGF-BB (50 ng/ml), and the culture was maintained for 72 hours. The exosomes from the culture supernatants were isolated with a routine ultra-catrifagation method. Flow cytometric analysis was performed to identify the origin of the exosomes, and their morphological features were observed by using a transmission electron microscopy. The exosomes were added at a concentration of 10 µg/ml into the culture system of human umbilical cord vein endothelial cells. MTT assay was used to evaluate the proliferative status. The Matrigel assay was used to observe the formation of net-work structures which were calculated after culture for 12 hours.</p><p><b>RESULTS</b>Flow cytometric analysis showed that microparticles released by human umbilical cord MSCs expressed CD9, CD63 and CD81, which was in accordance with the surface molecular features of exosomes. Under an electron microscope, the exosomes took the featured cystic shape. The protein contents of exosomes released by untreated, EPO-stimulated, PDGF-BB-stimulated and EPO plus PDGF-BB stimulated MSCs (10 cells) were 256±124 µg, 1021±392 µg, 830±265 µg and 2207±733 µg, respectively. The results revealed that MSCs treated by EPO and PDGF-BB released significantly higher amounts of exosomes (P<0.01). MTT assay proved that the exosomes from EPO and PDGF-BB treated MSCs had more potent proliferation-promoting activity on human umbilical cord vein endothelial cells than those from untreated MSCs. The Matrigel assay showed that the numbers of capillary-like structures in untreated, EPO-, PDGF-BB and EPO plus PDGF-BB-treated groups were 2.6±0.84, 4.6±1.57, 4.2±0.78 and 6.3±1.34 per high power objective. Treatment with EPO or PDGF-BB dramatically enhanced the numbers of capillary liue structure, compared with that of untreated group (P<0.01) and those in EPO and PDGF-BB combination group was significantly greater than those of EPO or PDGF-BB group (P<0.01).</p><p><b>CONCLUSION</b>EPO and PDGF-BB can stimulate MSCs to release exosomes with more potent proangiogenic activity.</p>
RESUMO
<p><b>OBJECTIVE</b>To observe the effect of pioglitazone on hypoxia/reoxygenation injury and the expression of protein kinase C (PKC) in neonatal rat cardiomyocytes.</p><p><b>METHODS</b>Neonatal Sprague-Dawley rat cardiomyocytes in primary culture were treated with pioglitazone or GW9662 for 24 h prior to hypoxia/reoxygenation injury. Cardiomyocyte apoptosis was evaluated with Hoechst33258 staining and the expression of PKC was detected using Western blotting.</p><p><b>RESULTS</b>In the early stage of hypoxia/reoxygenation injury, the apoptosis rates of the cardiomyocytes increased significantly from (0.20∓0.03)% of the control level to (12.22∓1.45)% (P<0.05). Pretreatment with pioglitazone significantly lowered the apoptosis rate of the cardiomyocytes with hypoxia/reoxygenation injury to (8.32∓0.89)%, and this effect was antagonized by GW9662, a specific blocker of peroxisome proliferators activated receptors γ (PPARγ). Pioglitazone did not cause increased expression of PKC in the cardiomyocytes.</p><p><b>CONCLUSION</b>Pioglitazone can ameliorate neonatal rat cardiomyocyte injury induced by hypoxia/reoxygenation partially by activating PPARγ and does not increase the expression of PKC in the cells.</p>