Doxorubicin (DOX) induces endothelial cell (EC) senescence, which contributes to endothelial dysfunction and cardiovascular complications. Senolytic drugs selectively eliminate senescent cells to ameliorate senescence-mediated pathologies. Previous studies have demonstrated differences between immortalized and primary EC models in some characteristics. However, the response of DOX-induced senescent ECs to senolytics has not been determined across these two models. In the present work, we first established a comparative characterization of DOX-induced senescence phenotypes in immortalized EA.hy926 endothelial-derived cells and primary human umbilical vein EC (HUVECs). Thereafter, we evaluated the senolytic activity of four senolytics across both ECs. Following the DOX treatment, both EA.hy926 and HUVECs shared similar senescence phenotypes characterized by upregulated senescence markers, increased SA-β-gal activity, cell cycle arrest, and elevated expression of the senescence-associated secretory phenotype (SASP). The potentially senolytic drugs dasatinib, quercetin, and fisetin demonstrated a lack of selectivity against DOX-induced senescent EA.hy926 cells and HUVECs. However, ABT-263 (Navitoclax) selectively induced the apoptosis of DOX-induced senescent HUVECs but not EA.hy926 cells. Mechanistically, DOX-treated EA.hy926 cells and HUVECs demonstrated differential expression levels of the BCL-2 family proteins. In conclusion, both EA.hy926 cells and HUVECs demonstrate similar DOX-induced senescence phenotypes but they respond differently to ABT-263, presumably due to the different expression levels of BCL-2 family proteins.

Sex is a salient risk factor in the development of doxorubicin-induced cardiotoxicity. Sex differences in the heart’s ability to respond to hypertrophic stimuli in doxorubicin-exposed animals have not been reported. We identified the sexual dimorphic effects of isoproterenol in mice pre-exposed to doxorubicin. Male and female intact or gonadectomized C57BL/6N mice underwent five weekly intraperitoneal injections of 4 mg/kg doxorubicin followed by a five-week recovery period. Fourteen days of subcutaneous isoproterenol injections (10 mg/kg/day) were administered after the recovery period. Echocardiography was used to assess heart function one and five weeks after the last doxorubicin injection and on the fourteenth day of isoproterenol treatment. Thereafter, mice were euthanized, and the hearts were weighed and processed for histopathology and gene expression analysis. Doxorubicin did not produce overt cardiac dysfunction in male or female mice before starting isoproterenol treatment. The chronotropic response to a single isoproterenol injection was blunted by doxorubicin, but the inotropic response was maintained in both males and females. Pre-exposure to doxorubicin caused cardiac atrophy in both control and isoproterenol-treated male mice but not in female mice. Counterintuitively, pre-exposure to doxorubicin abrogated isoproterenol-induced cardiac fibrosis. However, there were no sex differences in the expression of markers of pathological hypertrophy, fibrosis, or inflammation. Gonadectomy did not reverse the sexually dimorphic effects of doxorubicin. Additionally, pre-exposure to doxorubicin abrogated the hypertrophic response to isoproterenol in castrated male mice but not in ovariectomized female mice. Therefore, pre-exposure to doxorubicin caused male-specific cardiac atrophy that persisted after isoproterenol treatment, which could not be prevented by gonadectomy.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones located in atria granules. Both peptides respond to cardiac pressure and volume dynamics and accordingly serve as translation biomarkers for the clinical treatment of heart failure. Serum ANP and BNP play central secretary roles in blood pressure and cardiac output regulation and have proven utility as differential biomarkers of cardiovascular proficiency and drug-induced maladaptation, yet both peptides are impervious to exercise-induced hypertrophy. We employed immunoelectron microscopy to examine the effects of 28 days of chronic swim exercise or administration of a PPARγ agonist on atrial granules and their stored natriuretic peptides in Sprague Dawley rats. Chronic swimming and drug treatment both resulted in a 15% increase in heart weight compared with controls, with no treatment effects on perinuclear granule area in the left atria (LAs). Drug treatment resulted in larger size granules with greater BNP density in the right atria. Comparing swimming and PPARγ agonist treatment effects on ANP:BNP granule density ratios between atrial chambers revealed a shift toward a greater proportion of ANP than BNP in LAs of swim-trained rats. These data suggest a distinction in the population of ANP and BNP after chronic swim or PPARγ that makes it a novel metric for the differentiation of pathological and physiological hypertrophy.

ID 26880 Poster Board 357 Introduction: Recently, cancer survivors were shown to have a higher risk of severe infections that require hospitalization. Doxorubicin (DOX) is a widely used chemotherapeutic agent in the treatment of cancer patients. DOX has been shown to induce senescence phenotype in endothelial cells (ECs) characterized by endothelial dysfunction and secretion of pro-inflammatory cytokines termed senescence-associated secretory phenotype (SASP). Lipopolysaccharide (LPS), a bacterial component that stimulates inflammation, has been demonstrated to augment the inflammatory SASP phenotype in endothelial models of replicative senescence and irradiation-induced senescence. However, the effect of LPS in DOX-induced endothelial senescence has not been established. Therefore, in the current work, we determined the effect of LPS stimulation in DOX-induced senescent ECs in vitro. Then, we identified the effects of metformin, an anti-diabetic drug that has senomorphic properties, on LPS-induced hyperinflammation. Methods: Human umbilical vein endothelial cells (HUVECs) were either treated with DOX for 24 hours followed by 72 hour-incubation without DOX to establish senescence (Sen-HUVECs) or left untreated as non-senescent proliferating cells (NS-HUVECs). The effect of LPS was determined by stimulating both Sen-HUVECs and NS-HUVECs with LPS (30 ng/ml) for an additional 24 hours. The effects of metformin on LPS stimulation were assessed in both types of cells. Culture media were collected to measure SASP factors using ELISA. Protein expression of the senescence marker, p21, was measured to confirm the induction of senescence. To delineate the molecular mechanisms, the changes in NF-κB, a major signaling pathway involved in inflammation, were determined using western blotting. Results: DOX-induced senescence phenotype was demonstrated by upregulation of p21. A significant hyperstimulation response to LPS was observed in Sen-HUVECs compared to NS-HUVECs, demonstrated by higher secretion of SASP markers in the media including IL-6, CXCL2, and MMP3. The protein expression of ICAM-1, a marker of endothelial dysfunction, was also upregulated in Sen-HUVECs. Pretreatment with metformin blunted LPS-induced upregulation of SASP markers and normalized the expression of ICAM-1. Metformin effects were mediated by the downregulation of LPS-induced activation of NF-κB. Conclusions: Our current work shows that DOX-induced senescent endothelial cells show hyperinflammatory response to LPS. The senomorphic drug, metformin, can be a promising strategy to ameliorate infection-induced hyperinflammation in DOX-treated cancer survivors. Research was supported by the National Heart, Lung, and Blood Institute, grant R01HL151740. I.Y.A is supported by the Doctoral Dissertation Fellowship (DDF) offered by the University of Minnesota and the Bighley Graduate Fellowship from the College of Pharmacy.