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In endothelial cells, the tight control of the redox environment is essential for the maintenance of vascular homeostasis. The imbalance between ROS production and antioxidant response can induce endothelial dysfunction, the initial event of many cardiovascular diseases. Recent studies have revealed that the endoplasmic reticulum could be a new player in the promotion of the pro- or antioxidative pathways and that in such a modulation, the unfolded protein response (UPR) pathways play an essential role. The UPR consists of a set of conserved signalling pathways evolved to restore the proteostasis during protein misfolding within the endoplasmic reticulum. Although the first outcome of the UPR pathways is the promotion of an adaptive response, the persistent activation of UPR leads to increased oxidative stress and cell death. This molecular switch has been correlated to the onset or to the exacerbation of the endothelial dysfunction in cardiovascular diseases. In this review, we highlight the multiple chances of the UPR to induce or ameliorate oxidative disturbances and propose the UPR pathways as a new therapeutic target for the clinical management of endothelial dysfunction.

Background: Cystic fibrosis trans-membrane conductance regulator (CFTR) is a cAMP-modulated chloride/bicarbonate channel with different functions in various organs and tissues. Patients with CF are characterized by recurrent infections and by chronic neutrophilic airway inflammation as well as increased levels of neutrophil elastase (NE) in the airways. NE triggers cell cycle arrest that can lead to senescence. Moreover, it is well demonstrated that the CFTR expression get decreasing from embryogenesis to adult/elder age. Transcriptional, posttranscriptional and epigenetic regulatory mechanisms play a key role in decreasing the CFTR expression during life and recent evidences show that some CFTR-regulating microRNAs, i.e, mir-494, is up regulated during cell senescence. Cellular senescence is a permanent cell cycle arrest. In young cells it can be induced by different stimuli both exogenous and endogenous such as the physiological shortening of telomeres, the activation of oncogenes as well as oxidative stress. Furthermore, senescent cells are associated with modifications in the gene expression profile based on transcriptional, posttranscriptional and epigenetic regulatory mechanisms. Aims: To assess whether CFTR might be involved in cellular senescence and understand its role in the cellular senescence. Methods: To induce premature senescence, IMR90 cells at PDL34 were treated with 100 M diethylmaleate (DEM) (Sigma-Aldrich) in complete medium on alternate days for ten days. Western Blot and Real Time-PCR analysis were performed to assess either the CFTR and the microRNAs expression in: i) IMR90 cells at different population doubling levels (PDL); ii) IMR90 cells at different days after DEM treatment. Results:We found that: i) the CFTR expressions significantly decreased in IMR90 cells during replicative senescence (PDL58 versus PDL35); ii) the CFTR expression significantly decreased in IMR90 cells after DEM treatment; iii) there was an inverse relationship between CFTR and miRNA 494 expression either in IMR90 cells during replicative senescence and in IMR90 cells after DEM treatment. Conclusions: Our preliminary data show that CFTR is physiologically down-regulated during cellular senescence and this may (at least partially) depend on the up-regulation of miR-494. To better investigate the role of CFTR in cellular senescence, we are performing the over-expression of CFTR protein in pre-senescent/senescent IMR90 and the silencing of CFTR in IMR90 young cells. Furthermore, we are now validating such results on primary epithelial nasal cells from different subjects.

Neuroblastoma cells (NB) are able to counteract oxidative stress (OS) through the activation of antioxidant defense mechanisms among which Nrf2/HO-1 system plays a crucial role. By using SH-SY5Y NB cells in resting condition or after differentiation with all-trans retinoic acid (ATRA), we showed that differentiated cells are more sensitive to OS than undifferentiated cells, due to a reduction in the ability to upregulate HO -1. In order to clarify the molecular mechanisms underlying the impairment of HO-1 induction, Nrf2 and Bach1, have been analyzed. We showed that Nrf2 moves into the nucleus in response to H2O2 in both undifferentiated and differentiated cells. Instead, Bach1 expression decreased in undifferentiated cells exposed to H2O2 but no changes were observed in differentiated cells. Thus, we provided evidence that the lack of Bach1 degradation in differentiated cells increased sensitivity to OS. Moreover, bioinformatic analysis revealed that Bach1 is a putative target of miRNA494. We showed that NB differentiation down-regulates miR494 level. In addition, preliminary data show that miRNA494 inhibition in undifferentiated cells is able to reduce HO-1 induction in response to OS, increasing cell death.

We recently identified miR-494 up-regulation as a component of the program leading to cellular senescence of human diploid IMR90 fibroblasts [1]. To identify miR-494 targets, we used 2D-DIGE coupled to mass spectrometry analysis to profile protein changes induced by overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (p≤0.05) affecting a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction analysis for miR-494 binding sites on the relevant mRNAs, identified 26 putative targets of miR-494, with 7 of them featuring evolutionary conservation of miR-494 binding site. Functional miR-494 binding site were confirmed for hnRNPA3, PDIA3, RAD23B, and SYNCRIP. siRNA-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494, blunting cell proliferation and causing increase of SA-β-gal and DNA damage. Reintroduction of hnRNPA3 or RAD23B slowed the appearance of miR-494-induced senescent phenotype in IMR90 cells. Overall, these findings identify novel miR-494 direct targets involved in senescence. References: [1]Faraonio R et al. Cell Death Differ. 2012 Apr;19(4):713- 21. doi: 10.1038/cdd.2011.143. Epub 2011 Nov 4

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli such as telomere attrition, activated oncogenes, oxidative stress. We recently identified up-regulation of miR-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2D-DIGE coupled to mass spectrometry analysis to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (p≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction analysis of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational target prediction analysis identified 7 putative miR-494 targets, the 3’UTRs of which featured evolutionary conservation of binding sites for miR-494. Functional miR-494 binding sites were confirmed in 3’UTRs of four of them (hnRNPA3, PDIA3, RAD23B, and SYNCRIP/hnRNPQ). Accordingly, their reduced expression correlated with miR-494 up-regulation in different models of senescence. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-β-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494 in IMR90 cells. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.