Xiaodong Zheng

Objective: Circular RNAs are emerging as important regulators of pulmonary hypertension where pyroptosis plays a critical role. However, whether and how the circular RNAs regulate pyroptosis remained unexplored. Here, we show evidence for the involvement of a specific circular RNA known as circ-Calm4 in pulmonary hypertension and the underlying signaling pathway in pyroptosis. Approach and Results: Circ-Calm4 was upregulated in both mouse model of pulmonary hypertension in vivo and cultured smooth muscle cells in vitro. We performed immunoblotting, quantitative real-time PCR, LDH (lactate dehydrogenase) release assay, Annexin V-FITC/propidium iodide double staining, Hoechst 33342/propidium iodide fluorescence staining, and immunostaining to clarify the roles of circ-Calm4 in pulmonary arterial smooth muscle cell pyroptosis. Silencing the circ-Calm4 with its small-interfering RNA mitigated the upregulation of pyroptosis related phenotypes induced by hypoxia. Luciferase reporter assays confirmed that miR-124-3p suppressed the luciferase activity of the circ-Calm4 and RNA fluorescence in situ hybridization showed the colocalization of circ-Calm4 and miR-124-3p. The circ-Calm4 was found to act as a competitive endogenous RNA to regulate miR-124-3p. The pyroptosis-related alterations were all diminished with miR-124-3p in hypoxic pulmonary arterial smooth muscle cells. Inhibition of the gene targeted by miR-124-3p encoding the Pdcd6 (programmed cell death protein 6) abrogated pyroptosis-related phenotypes under hypoxia stimulation. Conclusions: Our findings show a new signaling pathway, the circ-Calm4/miR-124-3p/ Pdcd6 axis was demonstrated in regulation of hypoxia-induced pyroptosis, which may potentially be useful for the design of therapeutic strategies for protecting the cellular functionality against pyroptosis as well as pulmonary hypertension.

Reactive oxygen species (ROS) are involved in the pathologic process of pulmonary arterial hypertension as either mediators or inducers. Rutin is a type of flavonoid which exhibits significant scavenging properties on oxygen radicals both in vitro and in vivo. In this study, we proposed that rutin attenuated hypoxia-induced pulmonary artery smooth muscle cell (PASMC) proliferation by scavenging ROS. Immunofluorescence data showed that rutin decreased the production of ROS, which was mainly generated through mitochondria and NADPH oxidase 4 (Nox4) in pulmonary artery endothelial cells (PAECs). Western blot results provided further evidence on rutin increasing expression of Nox4 and hypoxia-inducible factor-1α (HIF-1α). Moreover, cell cycle analysis by flow cytometry indicated that proliferation of PASMCs triggered by hypoxia was also repressed by rutin. However, N-acetyl-L-cysteine (NAC), a scavenger of ROS, abolished or diminished the capability of rutin in repressing hypoxia-induced cell proliferation. These data suggest that rutin shows a potential benefit against the development of hypoxic pulmonary arterial hypertension by inhibiting ROS, subsequently preventing hypoxia-induced PASMC proliferation.