Abstract
Background: Pulmonary arterial hypertension (PAH) is a devastating, rapidly degenerating disease characterized by lung vascular cell proliferation/remodeling and elevated vascular pressure leading to right heart failure. We previously showed that BMP antagonist-Gremlin1 elicits pulmonary endothelial cell (EC) proliferation in response to hypoxia, a stimulus that recapitulates in vivo changes occurring in human PAH also leading to vascular cell proliferation and remodeling. NADPH oxidase-derived reactive oxygen species (ROS) purportedly play a critical role in PAH; yet the mechanisms by which they propagate the disease are scant. Other studies show that the cAMP response element protein (CREB) is activated by ROS and modulates cell proliferation. We postulated that Nox1-mediated CREB activation leads to Gremlin1 expression promoting human pulmonary arterial EC (HPAEC) proliferation. Method: HPAECs were subjected to 24 hrs hypoxia (1% O 2 ) vs. normoxia (21% O 2 ). Nox1, Nox2, Nox4, active CREB (pCREB), Gremlin1 and active Smads1/5/8 (pSmads) were evaluated by Western blot. Superoxide anion (O 2 •- ) changes were assessed using cytochrome C. To evaluate whether CREB binds to Gremlin1 promoter, chromatin immunoprecipitation (CHIP) was performed. Results: Hypoxia upregulated Nox1 (58% increase vs. normoxia, p<0.0001) but did not affect Nox2 and 4 levels. Hypoxia induced O 2 •- (13.6±1.5 vs. 18.3±0.6 nmol/min*mg, normoxia v. hypoxia, respectively, p<0.05). Furthermore, hypoxia increased pCREB (66% vs. normoxia, p<0.05), and Gremlin1 (39% increase vs. normoxia) whereas it decreased pSmads 1/5/8 (50% reduction vs. normoxia, p<0.05). Nox1 siRNA decreased pCREB (48% reduction vs. hypoxia alone, p=0.08). Finally, preliminary data show CREB binding to the Gremlin1 promoter. Discussion: In the present study, we found that hypoxia-induced HPAEC O 2 •- derived from Nox1 appears to mediate CREB activation, and subsequent promotion of Gremlin1 expression. Taken together, our data are consistent with CREB mediating Nox1-Gremlin1 signaling in hypoxia-induced EC proliferation.
Published Version
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