PURPL and NEAT1 Long Non-Coding RNAs Are Modulated in Vascular Smooth Muscle Cell Replicative Senescence.

Cellular senescence is characterized by proliferation and migration exhaustion, senescence-associated secretory phenotype (SASP), and oxidative stress. Senescent vascular smooth muscle cells (VSMCs) contribute to cardiovascular diseases and atherosclerotic plaque instability. Since there are no unanimously agreed senescence markers in human VSMCs, to improve our knowledge, we looked for new possible senescence markers. To this end, we first established and characterized a model of replicative senescence (RS) in human aortic VSMCs. Old cells displayed several established senescence-associated markers. They stained positive for the senescence-associated ß-galactosidase, showed a deranged proliferation rate, a dramatically reduced expression of PCNA, an altered migratory activity, increased levels of TP53 and cell-cycle inhibitors p21/p16, and accumulated in the G1 phase. Old cells showed an altered cellular and nuclear morphology, downregulation of the expression of LMNB1 and HMGB1, and increased expression of SASP molecules (IL1ß, IL6, IL8, and MMP3). In these senescent VSMCs, among a set of 12 manually selected long non-coding RNAs (lncRNAs), we detected significant upregulation of PURPL and NEAT1. We observed also, for the first time, increased levels of RRAD mRNA. The detection of modulated levels of RRAD, PURPL, and NEAT1 during VSMC senescence could be helpful for future studies on potential anti-aging factors.

Proximity Ligation Assay Detection of Protein-DNA Interactions-Is There a Link between Heme Oxygenase-1 and G-quadruplexes?

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1-/- mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4-protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.