Evidence that Cell free hemoglobin induces rat pulmonary artery smooth muscle cell proliferation by decreasing nuclear Cyclic AMP Response Element Binding (CREB) protein by a mechanism(s) other then oxidative stress

Clinical observations have demonstrated a high prevalence of pulmonary arterial hypertension (PH) in patients suffering from chronic hemolytic disease syndromes. It has been proposed that chronic exposure to cell free hemoglobin (CFH) contributes to the development of PH by either its nitric oxide‐scavenging or pro‐oxidant characteristics; however, important mechanistic studies on how these traits relate to pulmonary artery smooth muscle (PASMC) proliferation associated with PH have not yet occurred. Previous studies have shown that loss of nuclear Cyclic AMP Response Element Binding (CREB) protein is associated with PASMC proliferation. We hypothesized that CFH decreases the concentration of nuclear CREB, which then induces PASMC proliferation. Methods Quiescent rat pulmonary arterial smooth muscle cells (rPASMC) were incubated for 24h with three states of CFH (ferrous, ferric and ferryl) in the presence or absence of super oxide dismutase (SOD) and/or catalase (CAT). Colorimetric assay and cell counting were used to quantify the rate of cellular proliferation. In addition, we measured nuclear CREB activation by nuclear extraction and western blot analyzes. Results Compared to untreated cells, all forms of CFH decreased nuclear CREB. Yet, when rPASMC were treated with CFH and the ferryl state induced the most proliferation. This was true for both the colorimetric and cell counting techniques. This induction was not inhibited with the co‐treatment of SOD and/or CAT. Conclusion CFH can directly affect the proliferation rate of PASMC and concentration of nuclear CREB by a mechanism(s) other then oxidative stress