The Role Of Transforming Growth Factor β1 In Hypoxia Inducing Transdifferentiation Of Pulmonary Artery Endothelial Cells To Smooth Muscle-like Cells

Objective:To investigate the transdifferentiation of pulmonary artery endothelium cells to smooth muscle-like cells under hypoxia and the role of transforming growth factor 1 ( TGFβ1) in this progress.Methods1. Endothelial cells from pulmonary artery of pig were purified by immunomagnetic (Dynabeads) purification techniques(based on PECAM-1/CD31 positive expression for sorting marker). The obtained pulmonary artery endothelial cells(PAECs)were primary cultured and subcultured.2. Using the immunocytochemistry to detect the expression of factorⅧand CD31 together with morphological observation through inverted phase contrast microscope to determined the purified cells were PAECs.3. The purified endothelial cells were divided into four groups :①normoxia group (cell culture box containing 21% O2, 5%CO2 and 74% N2)②hypoxia group (cell culture box containing 1%O2,5%CO2,94%N2 ).③normoxia culturing using TGFβ1neutralizing Abs to block the expression of TGFβ1,④hypoxia culturing using TGFβ1neutralizing Abs to block the role of TGFβ1. four groups were cultured 1day, 4days and 7 days respectively.4. Using the immunocytochemistry to detect the expression of smooth muscle cells specific markerα-SM- actin together with morphological observation to determined whether the endothelial cells transdifferentiated to the smooth muscle-like cells or not.5. Using reverse transcription- polymerase chain reaction (RT-PCR) analysis the expression of TGFβ1 mRNA in normoxia group and hypoxia group6 The expression of TGFβ1 protein were analyzed by Western blot. Results:1. The MACS (magnetic cell separation system) can highly purify endothelial cells. The cells which were purified by immunomagnetic (Dynabeads) purification techniques formed typical monolayer of cobblestone appearance and identifed by factorⅧand CD31 positive immunohistochemical stain.2. Identification of smooth muscle-like cells: These cells which were spindle-shaped and expressed smooth muscle cells specific markerα-SM- actin were identified SMCs.3. With the hypoxic time prolongation, the ratio of transdifferentiated smooth muscle-like cells from pulmonary aterial endothelial cells was gradually increased (P<0.05); blocking the role of TGFβ1, however, could significantly decrease the ration of transdifferentiation (P<0.05).4. The expression level of TGFβ1 mRNA and protein was higher in hypoxia groups than that in nomoxia groups (P<0.05).Conclusions:1. The MACS is efficiency in endothelial cell isolation and purification and can obtain highly purified endothelial cell fast and easily.2. Present study shows that pulmonary arterial endothelial cells colony contains those cells which possess the transdifferentiation potential to smooth muscle-like cells.3.Hypoxia is greatly attributed to the transdifferentiation.4.TGFβ1 plays a important role in this process.