3-(2-4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3h)-quinazolinone and Hypertension--Pulmonary

N-myc downstream regulated gene 1 (NDRG1) has recently drawn increasing attention because of its involvement in angiogenesis, cell proliferation, and differentiation. We used in vitro [human pulmonary artery smooth muscle cells (hPASMCs)] and in vivo (rat) models under hypoxic conditions and found a vital role of NDRG1 in reducing apoptosis and increasing proliferation and migration by overexpressing and knocking down NDRG1. We also proved that hypoxia induced the protein expression of dynamin-related protein 1 (DRP1) and stimulated The phosphatidylinositol-3-kinase (PI3K)/ Protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathways, and these effects were reversed by NDRG1 knockdown. The relationship between NDRG1 and DRP1 and the PI3K/Akt/mTOR pathway was further evaluated by adding mdivi-1 (DRP1 inhibitor) or LY294002 (PI3K inhibitor). NDRG1 was found to regulate the proliferation, apoptosis, and migration of hypoxia-treated hPASMCs via DRP1 and PI3K/Akt/mTOR signaling pathways. We explored the upstream regulators of NDRG1 using in vivo and in vitro hypoxia models. Hypoxia was found to upregulate and downregulate KLF transcription factor 4 (KLF4) protein expression in the cytoplasm and nucleus, respectively. Further, we showed that KLF4 regulated the proliferation and migration of hypoxia-treated hPASMCs via NDRG1. These results indicated a link between KLF4, NDRG1, and DRP1 for the first time, providing new ideas for treating hypoxic pulmonary hypertension.

Topics: Animals; Cell Hypoxia; Dynamins; Humans; Hypertension, Pulmonary; Hypoxia; Mammals; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; TOR Serine-Threonine Kinases

Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction caused, in part, by pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1α (HIF-1α) have been observed in PAH PASMCs; however, their relationship and relevance to the development of PAH are unknown. Dynamin-related protein-1 (DRP1) is a GTPase that, when activated by kinases that phosphorylate serine 616, causes mitochondrial fission. It is, however, unknown whether mitochondrial fission is a prerequisite for proliferation.. We hypothesize that DRP1 activation is responsible for increased mitochondrial fission in PAH PASMCs and that DRP1 inhibition may slow proliferation and have therapeutic potential.. Experiments were conducted using human control and PAH lungs (n=5) and PASMCs in culture. Parallel experiments were performed in rat lung sections and PASMCs and in rodent PAH models induced by the HIF-1α activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1α activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1-dependent phosphorylation of DRP1 at serine 616. In normal PASMCs, HIF-1α activation by CoCl(2) or desferrioxamine causes DRP1-mediated fission. HIF-1α inhibition reduces DRP1 activation, prevents fission, and reduces PASMC proliferation. Both the DRP1 inhibitor Mdivi-1 and siDRP1 prevent mitotic fission and arrest PAH PASMCs at the G2/M interphase. Mdivi-1 is antiproliferative in human PAH PASMCs and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function, and hemodynamics in experimental PAH.. DRP-1-mediated mitotic fission is a cell-cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

Topics: Animals; Antihypertensive Agents; Case-Control Studies; CDC2 Protein Kinase; Cell Cycle Checkpoints; Cell Proliferation; Cells, Cultured; Cobalt; Cyclin B1; Disease Models, Animal; Dynamins; Enzyme Activation; Familial Primary Pulmonary Hypertension; Genetic Therapy; Glycolysis; GTP Phosphohydrolases; Humans; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Microtubule-Associated Proteins; Mitochondria, Muscle; Mitochondrial Proteins; Mitosis; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Pulmonary Artery; Quinazolinones; Rats; Rats, Sprague-Dawley; RNA Interference; Serine; Time Factors; Transfection