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Sublethal cell damage can trigger a complex adaptive program known as senescence, characterized by growth arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP). As senescent cells accumulating in aging organs are linked to many age-associated diseases, senotherapeutic strategies are actively sought to eliminate them. Here, a whole-genome CRISPR knockout screen revealed that proteins in the YAP-TEAD pathway influenced senescent cell viability. Accordingly, treating senescent cells with a drug that inhibited this pathway, Verteporfin (VPF), selectively triggered apoptotic cell death and derepressed DDIT4, in turn inhibiting mTOR. Reducing mTOR function in senescent cells diminished endoplasmic reticulum (ER) biogenesis, causing ER stress and apoptosis due to high demands on ER function by the SASP. Importantly, VPF treatment decreased senescent cell numbers in the organs of old mice and mice exhibiting doxorubicin-induced senescence. We present a novel senolytic strategy that eliminates senescent cells by hindering ER activity required for SASP production.

Cellular senescence is characterized by cell cycle arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP) whereby cells secrete pro-inflammatory and tissue-remodeling factors. Given that the SASP exacerbates age-associated pathologies, some aging interventions aim at selectively eliminating senescent cells. In this study, a drug library screen uncovered TrkB (NTRK2) inhibitors selectively capable of triggering apoptosis of senescent, but not proliferating, human fibroblasts. Senescent cells expressed high levels of TrkB, which supported senescent cell viability, and secreted the TrkB ligand BDNF. The reduced viability of senescent cells after ablating BDNF signaling suggested an autocrine function for TrkB and BDNF, including increased expression of BCL2L2 downstream of BDNF-TrkB, favoring senescent cell survival. Strikingly, treatment with TrkB inhibitors reduced the accumulation of senescent cells in aged mouse organs. We propose that the activation of TrkB by SASP factor BDNF promotes cell survival and could be exploited therapeutically to reduce the senescent-cell burden.

Introduction: The transformation of vascular smooth muscle cells (VSMC) into foam cells leading to increased plaque size and decreased stability is a key, yet understudied step in atherogenesis. We reported that Interleukin-19 (IL-19), a novel, anti-inflammatory cytokine, attenuates atherosclerosis by anti-inflammatory effects on VSMC. We tested the hypothesis that one mechanism was reduction in VSMC foam cell formation. Methods and Results: In this work we report that IL-19 induces expression of miR133a, a muscle-specific miRNA, in VSMC. Although previously unreported, we show that miR133a can target and reduce mRNA abundance, mRNA stability, and protein expression of Low Density Lipoprotein Receptor Adaptor Protein 1, (LDLRAP1), an adaptor protein which functions to internalize the LDL receptor. Mutations in this gene lead to LDL receptor malfunction and cause the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans. We also show that IL-19 reduces lipid accumulation in VSMC, as well as LDLRAP1 expression and oxLDL uptake in a miR133a-dependent mechanism. We show that LDLRAP1 is expressed in plaque and neointimal VSMC of mouse and human injured arteries. Transfection of miR133a and LDLRAP1 siRNA into VSMC reduces their proliferation and uptake of oxLDL. miR133a is significantly increased in plasma from hyperlipidemic compared with normolipidemic patients. Summary and conclusions: miR133a targets LDLRAP1 3’UTR and reduces its expression. Expression of miR133a in IL-19 stimulated VSMC represents a previously unrecognized link between vascular lipid metabolism and inflammation, and may represent a therapeutic opportunity to combat vascular inflammatory diseases.

Vascular smooth muscle cells (VSMC) play a critical role in the etiology and progression of many vascular diseases including atherosclerosis and restenosis. Our laboratory has found that one anti-inflammatory interleukin, IL-19, is atheroprotective and can decrease vascular inflammation by reduction in mRNA stability of inflammatory transcripts by reduction of activity of HuR, an mRNA stability protein. HuR translocates from the nucleus to the cytoplasm where it recognizes AU-rich elements present almost exclusively in the 3’UTR of pro-inflammatory genes. Proteins and pathways which limit HuR translocation are understudied, but may reduce inflammatory mRNA stability. Using MASS SPEC to identify HuR-interacting proteins under different inflammatory conditions, we identified one protein, Fragile X-related protein (FXR1), which interacts with HuR in inflammatory, but not basal conditions, a novel interaction. FXR1 mRNA expression is enhanced in muscle cells, but nothing has been reported on expression of FXR1 in VSMC or function for FXR1 in vascular disease. The FXR1 promoter contains multiple cholesterol-response elements, and in this study we demonstrate that FXR1 expression is increased in injured arteries and TNFα and oxLDL stimulated human VSMC, but also by IL-19. RNA EMSA demonstrates that FXR1 directly interacts with ARE in 3’UTR. SiRNA knock down of FXR1 in VSMC increases stability of inflammatory mRNA and protein abundance as well as VSMC proliferation, while overexpression of FXR1 reduces both their abundance and stability in addition to reducing proliferation. Since FXR1 appears to be a novel repressor of inflammatory proteins, and is also induced by IL-19, our overall hypothesis is that FXR1 expression and HuR interaction is an inflammation responsive, counter-regulatory mechanism to reduce abundance of pro-inflammatory proteins and therefore reduce inflammation.

Chronic administration of estrogen to the Fischer 344 (F344) rat induces growth of large, hemorrhagic pituitary tumors. Ten weeks of diethylstilbestrol (DES) treatment caused female F344 rat pituitaries to grow to an average of 109.2 +/- 6.3 mg (mean +/- SE) versus 11.3 +/- 1.4 mg for untreated rats, and to become highly hemorrhagic. The same DES treatment produced no significant growth (8.9 +/- 0.5 mg for treated females versus 8.7 +/- 1.1 for untreated females) or morphological changes in Brown Norway (BN) rat pituitaries. An F1 hybrid of F344 and BN exhibited significant pituitary growth after 10 weeks of DES treatment with an average mass of 26.3 +/- 0.7 mg compared with 8.6 +/- 0.9 mg for untreated rats. Surprisingly, the F1 hybrid tumors were not hemorrhagic and had hemoglobin content and outward appearance identical to that of BN. Expression of both growth and morphological changes is due to multiple genes. However, while DES-induced pituitary growth exhibited quantitative, additive inheritance, the hemorrhagic phenotype exhibited recessive, epistatic inheritance. Only 5 of the 160 F2 pituitaries exhibited the hemorrhagic phenotype; 36 of the 160 F2 pituitaries were in the F344 range of mass, but 31 of these were not hemorrhagic, indicating that the hemorrhagic phenotype is not merely a consequence of extensive growth. The hemorrhagic F2 pituitaries were all among the most massive, indicating that some of the genes regulate both phenotypes.