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Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Fund for Scientific Research (FWO) Flanders INSPIRE project (H2020-MSCA-ITN program) Background The chemotherapeutic doxorubicin (DOX) is frequently used to treat a wide variety of cancers, but the cardiotoxic side effects limits its clinical use in some patients. Additionally, DOX contributes to vascular toxicity, which may be an early manifestation of DOX-associated toxicity. Therefore, there is an interest to evaluate vascular function in patients receiving DOX-based treatment regimens. Purpose We aimed to assess arterial stiffness and endothelial dysfunction as potential markers of vascular toxicity in both DOX-treated cancer patients and a murine model. Methods Female breast cancer patients with a need for adjuvant or neoadjuvant DOX-based treatment were prospectively included. Chemotherapeutic regimen consisted of 12 weekly cycles of taxane treatment, followed by 4 cycles of DOX (and cyclophosphamide) treatment. Vascular function (endothelial function (FMD and RHI), arterial stiffness (Aix and cfPWV) and cardiac function (LVEF, hsTnI, NT-proBNP) were performed at baseline (T1), after completion of taxane treatment (T2) and after completion of DOX treatment (T3). In addition to the clinical study, male C57Bl6/J mice were intraperitoneally injected with 2 mg /kg (low dose) or 4 mg/kg DOX (high dose) once per week for 6 weeks. Arterial stiffness was assessed in vivo by measuring abdominal aorta pulse wave velocity (aaPWV) by high-frequency ultrasound imaging combined with ex vivo vascular function evaluation. Results Twenty patients (mean age 51.2 +/- 10.1 yrs) treated with DOX were included. After DOX treatment (T3 vs T2), left ventricular ejection fraction (LVEF) was decreased, while hsTnI and NT-proBNP levels were increased, indicating cardiotoxicity (Table 1). Likewise, LVEF was reduced in DOX-treated mice after 3 weeks, which persisted until the end of the treatment (6 weeks). In patients, RHI was impaired at T2 and T3 compared to T1, which indicates progressive endothelial dysfunction during treatment (Table 1). Consistent with these findings, DOX treatment in mice resulted in endothelial dysfunction, as evidenced by a lower basal nitric oxide (NO) index and reduced acetylcholine-induced endothelium-dependent vasorelaxation (Figure 1B & 1C). Finally, cfPWV was decreased at T2 and T3 compared to T1, even after correction for BP, suggesting rather an improvement of arterial stiffness (Table 1). Although DOX treatment in mice resulted in a similar trend towards lower aaPWV at the end of treatment (6 weeks), we observed an initial increase in aaPWV after 2 weeks (Figure 1A). Conclusion Collectively, these findings suggest that, apart from cardiotoxicity, DOX treatment results in early and consistent endothelial dysfunction, while evaluation of arterial stiffness may be time-sensitive. Hence, endothelial dysfunction may be a more reliable and sensitive marker than arterial stiffness to evaluate DOX-induced vascular toxicity in patients. Open in new tab Download slide Table 1 Open in new tab Download slide Figure 1 [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fonds voor Wetenschappelijk Onderzoek (FWO) Introduction Necroptosis is a form of regulated necrosis mediated by the kinase activity of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and executed by RIPK3 and mixed lineage kinase domain like pseudokinase (MLKL). Because necroptosis is involved in necrotic core development in atherosclerotic plaques, we investigated the effect of a RIPK1 S25D/S25D mutation, which prevents the activation of RIPK1 kinase, on atherogenesis in ApoE-/- mice. Furthermore, we pharmacologically inhibited RIPK1 kinase activity by administering GSK'547 to ApoE-/- Fbn1 C1039G+/- mice, a mouse model of advanced atherosclerosis. Purpose We aimed to study the effect of RIPK1 kinase inhibition on atherogenesis through a genetic and a pharmacological approach in ApoE-/- and ApoE-/- Fbn1 C1039G+/- mice, respectively. Methods ApoE-/- and ApoE-/- Fbn1 C1039G+/- mice were fed a Western-type diet (WD) for up to 24 weeks and plaque necroptosis was confirmed. ApoE-/- Ripk1+/+ (n=16) and ApoE-/- Ripk1 S25D/S25D (n=12) mice were fed a WD for 16 weeks to induce plaque formation. ApoE-/- Fbn1 C1039G+/- mice received WD supplemented with GSK'547 (10 mg/kg BW/day, n=12-13/group) for 20 weeks to evaluate the effect of pharmacological RIPK1 kinase inhibition on atherogenesis. Results After 16 weeks WD, atherosclerotic plaques of ApoE-/- Ripk1 S25D/S25D mice were significantly larger as compared to ApoE-/- Ripk1+/+ mice (167±34 vs. 69±18 103 µm2, P=0.01). Absolute cell numbers (350±34 vs. 154±33 nuclei) and deposition of glycosaminoglycans (Alcian blue: 31±6 vs. 14±4%, P=0.023) were increased in plaques from ApoE-/- Ripk1 S25D/S25D mice while macrophage content (Mac3: 2.3±0.4 vs. 9.8±2.4%, P=0.012) was significantly decreased. Increased deposition of glycosaminoglycans was also observed in the vessel wall of ApoE-/- Ripk1 S25D/S25D mice (Alcian blue: 4.3±1.5 vs. 1.4±0.4 %, P=0.04). Together with increased internal elastic lamina area and increased vessel wall thickness, this points towards hypertrophic positive vascular remodeling in ApoE-/- Ripk1 S25D/S25D mice. Plaque apoptosis was not different between both groups. In contrast, pharmacological inhibition of RIPK1 kinase with GSK'547 in ApoE-/- Fbn1 C1039G+/- mice did not significantly alter plaque size and vessel properties after 20 weeks WD, but induced plaque apoptosis (TUNEL: 124±17 vs. 68±8 cells/mm2, P=0.018; cleaved caspase 3: 0.49±0.13 vs. 0.14±0.04 %; p=0.021). Conclusion Inhibition of RIPK1 kinase activity in ApoE-/- Ripk1 S25D/S25D mice accelerated plaque progression and induced positive vascular remodeling. These effects were not observed after pharmacological inhibition with GSK'547 in ApoE-/- Fbn1 C1039G+/- mice. However, a switch to apoptosis was observed after GSK'547 treatment, stressing the complex involvement of RIPK1 in cell survival and death. [ABSTRACT FROM AUTHOR]