Tadalafil, a phosphodiesterase inhibitor protects stem cells over longer period against hypoxia/reoxygenation injury through STAT3/PKG-I signaling

Pharmacological preconditioning (PC) with tadalafil, a PDE5A inhibitor, enhances protein kinase G-1 (PKG-I) activity, resulting in stem cell survival. Protection by PC had two different phases, early (2 h) and late (24 h). However, the mechanism of protection during these phases remained grossly unknown. Mesenchymal stem cells (MSCs) from adult male Fischer-344 rats were cultured and pretreated with tadalafil (100 μM) for an hour and subjected to 2 h of hypoxia (1% O₂), followed by reoxygenation (HR: in vitro model mimicking ischemia/reperfusion). We observed (i) increased MSC survival with reduced cell cytotoxicity as revealed by low lactate dehydrogenase release and trypan blue staining, respectively, in tadalafil-treated cells upon HR; (ii) decrease in TUNEL positivity as well as caspase activity; (iii) an increase in pAkt/Akt, iNOS, eNOS, and pGSK3β/GSK3β during the early protection phase of PC, and this protection seemed to be a spontaneous adaptive response of MSCs against HR and was independent of tadalafil, whereas an increase in Bcl2/Bax was tadalafil dependent; and (iv) during the late phase, we observed phosphorylation of STAT3 at serine727, leading to its entry inside the nucleus and binding onto the promoter of PKG-I by three-fold (P<0.05). In conclusion, an increase in Bcl2/Bax during the early phase and transcriptional upregulation of PKG-I by STAT3 during the late phase were responsible for stem cell protection by tadalafil against ischemic injury.