Impridone enhances vascular relaxation via FOXO1 pathway

Kristeen R. McSweeney1, Laura K. Gadanec1, Tawar Qaradakhi1, Thushira Malindra Gammune 1, Peter Kubatka2, Martin Caprnda3, Julia Fedotova4,5,6, Jozef Radonak7, Peter Kruzliak8, Anthony Zulli1

1Institute for Health and Sport, Victoria University, Melbourne, VIC, Australia
2Department of Medical Biology, Jessenius Faculty of Medicine, Comenius University in Bratislava, Martin, Slovakia
31st Department of Internal Medicine, Faculty of Medicine and University Hospital, Bratislava, Slovakia
4Institute of Biology and Biomedicine, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russian Federation
5International Research Centre “Biotechnologies of the Third Millennium”, ITMO University, St. Petersburg, Russian Federation
6Laboratory of Neuroendocrinology, I.P. Pavlov Institute of Physiology, Academy of Sciences, St. Petersburg, Russian Federation
71st Department of Surgery, Faculty of Medicine, Pavol Jozef Safarik University and University Hospital, Kosice, Slovak Republic
82nd Department of Surgery, Faculty of Medicine, Masaryk University and St. Anne´s University Hospital, Brno, Czech Republic
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1440-1681.13377

Corresponding authors:

Dr. Peter Kruzliak, 2nd Department of Surgery, Faculty of Medicine, Masaryk University and St. Anne´s University Hospital, Pekarska 53, 65691 Brno, Czech Republic, tel. +420 543 181 111, e- mail: [email protected]

Dr. Anthony Zulli, Institute for Health and Sport, Victoria University, Melbourne, VIC, Australia, [email protected]

Dr. Jozef Radonak, 1st Department of Surgery, Faculty of Medicine, Pavol Jozef Safarik University and University Hospital, Tr. SNP 1, 041 66 Kosice, Slovak Republic, e-mail: [email protected]


Cardiovascular complications are a side effect of cancer therapy, potentially through reduced blood vessel function. ONC201(TIC10) is currently used in Phase 2 clinical trials to treat high- grade gliomas. TIC10 is a Phosphatidylinositol 3-kinase (PI3K)/AKT/extracellular signal- regulated kinase (ERK) inhibitor that induces apoptosis via upregulation of TNF-related apoptosis-inducing ligand, which via stimulation of FOXO and death receptor could increase eNOS upregulation. This has the potential to improve vascular function through increased NO bioavailability. Our aim was to investigate the role of TIC10 on vascular function to determine if it would affect the risk of CVD. Excised Abdominal aorta from White New Zealand male rabbits were cut into rings. Vessels were incubated with TIC10 and AS1842856 (FOXO1 inhibitor) followed by cumulative doses of acetylcholine (Ach) to assess vessel function. Vessels were then processed for immunohistochemistry. Incubation of blood vessels with TIC10 resulted in enhanced vasodilatory capacity. Combination treatment with the FOXO1 inhibitor and TIC10 resulted in reduced vascular function compared to control. Immunohistochemical analysis indicated a 3-fold increase in death receptor 5 (DR5) expression in the TIC10 treated blood vessels but the addition of the FOXO1 inhibitor downregulated DR5 expression. The expression of DR4 receptor was not significantly increased in the presence of TIC10, however, addition of the FOXO1 inhibitor downregulated expression. TIC10 has the capacity to improve the function of healthy vessels when stimulated with the vasodilator Ach. This highlights its therapeutic potential

not only in cancer treatment without cardiovascular side effects, but also as a possible drug to treat established CVD.
Key Words: Cardiovascular Diseases; Vascular Relaxation; Impridone (TIC10); Nitric Oxide; Acetylcholine; FOXO1 pathway


Cardiovascular disease (CVD) is one of the leading causes of morbidity and mortality, both globally and in Australia.1 Atherosclerosis has long been implicated as one of the primary causes of CVD resulting from the dislodgement of atherosclerotic plaque.2 Atherosclerosis has been attributed as the leading cause of myocardial infarction and strokes, with approximately 50% of deaths in westernized society associated with atherosclerosis.3
Endothelial dysfunction has been established as a precursor to the development of atherosclerosis and has thus become a considerably targeted area for recent cardiovascular research.4
The endothelium is involved in many cellular processes including; inhibition of Vascular Smooth Muscle Cell (VSMC) proliferation, regulation of blood flow and vascular tone, prevention of leukocyte and platelet adhesion/aggregation and inflammation.5,6 It plays a homeostatic role in maintaining the balance between endothelium derived relaxation and constriction factors. Dysregulation of Nitric Oxide (NO) and altered redox states are two key factors associated with endothelial dysfunction.7 Nitric Oxide (NO) is an endothelium dependent and produced vasodilator. The activation and activity of Endothelial Nitric Oxide Synthase (eNOS) a fundamental catalyst for NO production is essential in ensuring endothelial function. Reduction in the bioavailability of NO has been associated with diseases relating to the cardiovascular system, such as hypertension and atherosclerosis.8 Discovery of new pharmaceutical approaches are crucial for the treatment and prevention of atherosclerotic risk factors such as endothelial dysfunction.

TIC10 is an imipridone, a novel class anticancer agent that’s clinical and experimental application to date has been limited to various cancer cell types.9-12 Its anticancer mechanism is well established within the literature. TIC10 results in the dual inhibition of Akt and ERK signalling pathways, causing dephosphorylation and nuclear translocation of the transcription factor FOXO3a enabling the upregulation of Tumour necrosis factor-related apoptosis inducing ligand

(TRAIL) gene transcription.9 TRAIL is a mediator of apoptosis via caspase-8 mediated apoptosis, however there are studies suggesting a pro-survival pathway for TRAIL.13 TRAIL has been linked to the attenuation of atherosclerosis in apolipoprotein E deficient mice.14 Although the mechanism of TIC10 in cancer cells is progressing, its physiological mechanism in endothelial cells is less known. Henceforth, understanding the role of TIC10 in endothelial cells may highlight its potential role in the prevention of risk factors associated with atherosclerosis and other vascular diseases. Many studies have investigated the function of Human Umbilical Vein Endothelial cells (HUVECs) when incubated with recombinant TRAIL however investigating the effects of the TRAIL inducing drug TIC10 on endothelial cells is yet to be investigated.

The FOXO family are a group of forkhead box proteins that are regulated by the PI3K/AKT signalling pathway.15 The FOXO family are transcription factors that play roles in cell survival through Akt phosphorylation, inhibiting the transcriptional function of FOXO.16 Of the FOXO family isoforms, studies have established that FOXO1 and FOXO3a are the most abundant in endothelial cells,17 with FOXO1 playing a large role in vascular formation, with its absence proving embryonically lethal at E10.5 in mice18. FOXO family proteins are involved in the upregulation of death receptor expression and are considered key modulators of apoptotic cell death.19 The extrinsic apoptotic pathway initiates the caspase cascade stimulated by ligand binding of cell surface death receptors DR4 and DR5.20 The aim of this study was to investigate the effects of TIC10 pre-incubation on vascular function in-vitro. Further to this it was investigated whether FOXO1 inhibition following treatment with TIC10 influenced vascular function and the expression of death receptors.


Acetylcholine mediated vascular relaxation

Blood vessels pre-incubated with 5mM TIC10 resulted in increased vascular relaxation compared to control after stimulation with Ach. Statistically the relaxation was significant at Ach concentrations 10-7 M -76.18.7% vs -61.75.22 % p<0.05) and 10-6.5 M (-767.8% vs - 92.64.6% p<0.05). Incubation with a FOXO1 inhibitor worsened acetylcholine stimulated

vascular relaxation compared to healthy vessels at ach concentrations 10-7 M (-61.75.2% vs - 42.110.7%) and 10-5 M ( -95.81.3% vs -77.23.3%) (Figure 1).
The addition of a FOXO1 inhibitor concurrently with TIC10 treatment had no effect on the enhancement on vascular function compared to the singular treatment with the FOXO1 inhibitor. The pre-incubation of vessels with a FOXO1 inhibitor followed by TIC10 treatment
resulted in reduced vascular relaxation compared to TIC10 treatment only, at ach doses 10-6.5 M (- 92.6  4.6% vs -60.1  6.8% p<0.05), 10-6 M (-97.3  3.8% vs -74.4  4.2% p<0.05) and 10-5.5 M (-99.5  3.1% vs -80.6  2.5% p<0.05) (Figure 1).
Significant vascular relaxation in the control vs FOXO1 inhibitor + TIC10 treated groups was observed at ach doses 10-7 M (-61.6  5.2% vs -28.9  5.2% p<0.05), 10-5.5 M (-93.1  0.3% vs - 80.6  2.5% p<0.05) and 10-5 M (-95.8  1.3% vs -79.8  3.4% p<0.05) (Figure 1).

Area under the curve graph of relaxation results obtained in Figure 1 highlighting significances between groups obtained via one-way ANOVA and unpaired t test.

Analysis of the area under the curve of figure 1 demonstrated significance between the following groups obtained via one way ANOVA: Control vs. FOXO1 Inhibitor + TIC10 (194.96.2 vs 144.110.3, P=0.01), TIC10 vs. FOXO1 Inhibitor (224.78.2 vs 17610, P=0.009) and TIC10 vs. FOXO1 Inhibitor + TIC10 (224.78.2 vs 144.110.3, P=0.0005). Significance was obtained in Control vs TIC10 treated group using unpaired t test (194.96.2 vs 224.78.241, P=0.04) (Figure 2).

Up-regulation of endothelial DR5, but not DR4 in response to TIC10 treatment
Treatment of abdominal aorta with TIC10 resulted in elevated expression of DR5 when compared to control (3.9±0.8 vs. 1±0.2, p<0.05). Vessels were pre-incubated with a FOXO1 inhibitor to investigate if FOXO1 plays a role in the vasorelaxation effects exhibited by TIC10.Inhibition of FOXO1 reduced the DR5 upregulation effects of TIC10 (1.1±0.2% vs. 3.9±0.8%, p<0.05) (Figure 3, Figure 4).

Downregulation of endothelial DR4 in the presence of a FOXO1 inhibitor
Addition of a FOXO1 inhibitor of abdominal aorta resulted in a significant reduction in endothelial DR4 expression compared to control (0.37±0.07 vs. 1±0.19, p<0.05) (Figure 5). The addition of TIC10 had no effect on the downregulation effects exerted by the FOXO1 inhibitor (Figure 5, Figure 6).


This study showed that TIC10 has a positive effect on acetylcholine induced aortic relaxation, and that this effect can be blocked by inhibiting FOXO1. The relationship between FOXO1 and FOXO3a remains unclear, with a large proportion dedicated to isolation studies investigating the roles each have in oncology research. A study investigating the role of TIC10 on colon cancer cells investigated TRAIL upregulation. It was determined that TRAIL upregulation induced by TIC10 was FOXO3a dependent, a finding that has been repeated extensively in oncology research.21-23 They showed that FOXO1 was not translocated into the nucleus, however FOXO3a was, enabling stimulation of TRAIL transcription. This nuclear translocation of FOXO3a was also accompanied by an increase in DR5 expression in these cancer cells. The anticancer properties exhibited by TIC10 were also abolished when FOXO3a activity was inhibited.9 Although there is a clear link between FOXO3a and TRAIL in both cancer cells and endothelial cells,12,17 little research has looked at the interrelationship between FOXO3a and FOXO1 and TRAIL and FOXO1, in any cell type.
A study in LAPC4 prostate carcinoma cells, investigated the effects of FOXO3a and FOXO1 on TRAIL gene transcription. The results of their study concluded that both FOXO3a and FOXO1 can regulate TRAIL transcription, with FOXO3a also found to induce FOXO1 expression.21 As a result of this our study aimed to investigate the role of FOXO1 on death receptor upregulation in the endothelium. A study investigating the role of FOXO1 in the vasculature showed that glucose induced oxidative stress resulted in FOXO1 activation in a hyperglycaemic model of atherosclerosis. The results illustrated a proatherogenic effect on endothelial cell function.24
A study in HEK293T and MEF cells showed that FOXO3a stimulates the expression of FOXO1 mRNA and it is suggested that FOXO3a induces autophagy via FOXO1 upregulation. The same study also showed that FOXO3a requires FOXO1 to elicit transcription-dependent autophagy.25 It has also been demonstrated that overexpression of FOXO1 leads to TRAIL upregulation in

cardiomyocytes.26 As described, there are several interrelationships between the functions of FOXO3a and FOXO1 in a variety of different cell types, potentially linked to their structural similarities, given often one is required for the other to elicit certain functions. This may be the case observed throughout this study.

TIC10 is well established in cancer cells to result in TRAIL upregulation resulting in activation of the extrinsic apoptosis pathway.11,27 TRAIL is the only known ligand that binds to DR4 and DR5.20 Immunohistochemical staining illustrated the upregulation of endothelial DR5 (P<0.05) in abdominal aorta incubated with TIC10, shown in figure 4. This indicates that in addition to DR5 expression, TRAIL transcription is also likely increased. Studies early on after the discovery of TIC10 and TRAIL showed that TRAIL had high affinity binding amongst all five of its receptors, making it difficult to understand the individual roles of the receptors that are recognized by the same ligand. Further research undertaken by Truneh and colleagues were able to use isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay to demonstrate that TRAIL had different affinities for each receptor at varying physiological temperatures.28 At 4 C, four of the five TRAIL receptors share equal binding affinity, however, at 37 C (the normal human physiological body temperature) DR5 had the highest affinity. Given the results obtained in Trunehs study this could explain why in this study, DR5 expression was significantly increased in the TIC10 treated group compared to control and why there was no upregulation of endothelial DR4 in response to TIC10 treatment. Literature suggests that at 37 C there is a higher affinity for DR5, and therefore it would be expected that there would be a higher expression of DR5 comparable to DR4, which was the case in this study. Although these results align with others who have investigated the specific role TRAIL plays, it cannot be concluded that other unknown ligands that could be causing upregulation of the death receptors.

It is plausible that the increased relaxation is a result of TIC10 stimulated TRAIL transcription. A study in HUVEC cells treated with recombinant TRAIL exerted protective effects on the vasculature via Akt-dependent eNOS signalling. Endothelial dysfunction is linked to a reduction in NO bioavailability. Increased eNOS phosphorylation and activity has been linked to improved vascular function through increased NO bioavailability. This is the mechanism we propose TIC10 is stimulating to exert its vasoprotective effects.

Many studies have used pharmacological interventions to investigate their mechanisms on eNOS and consequential endothelial function and cardio protection. However, increased eNOS expression might not correlate with elevated NO production. Resveratrol is an activator of sirtuin
1(SIRT1) an NAD+ dependent protein deacetylase29 and is involved in the SIRT1-FOXO1 signalling axis. A study undertaken in 2013 was used to investigate the mechanistic role of resveratrol on eNOS transcription. Resveratrol is a phytoalexin that has been linked to reduced prevalence of myocardial infarction in European countries along with other cardioprotective outcomes, postulated to be a result of increased NO bioavailability.30 Permanent human cell lines (EA.hy926) were treated with Resveratrol. The results demonstrate that eNOS expression was upregulated in a concentration dependent manner. It was established that not only was eNOS expression upregulated, eNOS enzymatic activity was also increased.30 The FOXO family are downstream targets of SIRT1 signalling, with treatment of EA. hy926 cells with resveratrol resulting in elevated expression of both FOXO1 and FOXO3a. SIRT1 knockdown by SiRNA was undertaken to silence FOXO1 and FOXO3a which resulted in reduced expression of eNOS. This could explain how the addition of the FOXO1 inhibitor reduced the vasorelaxation effects of control vessels at ach concentrations 10-7 and 10-5M. Given that FOXO3a is also known to upregulate both TRAIL and eNOS activity it is possible that the FOXO1 inhibitor repressed the transcriptional activity of FOXO3a in addition to FOXO1 consequently reducing eNOS activity. There are additional studies that have demonstrated that SIRT1 does not completely inhibit FOXO1 activity and implicated other cofactors in the regulation of FOXO1 functions.31 SIRT1 however was not the inhibitor used in this study however the inhibitor used here may be exerting the same functions. The reduction in eNOS when FOXO1 and FOXO3a were inhibited thereby likely reduced the production and bioavailability of freely diffusible NO preventing relaxation of Vascular Smooth Muscle Cells (VSMCs) of the FOXO1 inhibitor group presenting a possible explanation as to the reduction in vascular relaxation of FOXO1 inhibitor treated vessels as presented in this study. There was no significant difference in relaxation between the vessels treated with just the FOXO1 inhibitor and those treated with the FOXO1 inhibitor adjunctly with TIC10. These results indicate that FOXO1 plays a role in the vasodilatory response of vessels incubated with TIC10.
Interestingly, FOXO1 inhibition prevented the increase in DR5 upregulation. This could be due to a reduction in TRAIL transcription, indicating that FOXO1 plays a role in TIC10 stimulated DR5 upregulation. Literature suggests that Akt is a downstream target for both FOXO1 and FOXO3a

and therefore the ability of TIC10 to inactivate Akt maybe enabling the translocation of both FOXO1 and FOXO3a to stimulate TRAIL transcription and DR mediated apoptosis. However, the prevention of increased death receptor expression in the presence of a FOXO1 inhibitor indicates FOXO1 and FOXO3a either independently or synergistically stimulates TRAIL upregulation. This suggests that inhibition of FOXO1 results in the inhibition of FOXO3a. Further investigations need to be undertaken to investigate the relationship and crosstalk between FOXO1 and FOXO3a in endothelial cells and the effect it has on TRAIL transcription and expression.

Treatment of vessels with the FOXO1 inhibitor followed by incubation with TIC10 worsened Ach-dependent vascular relaxation compared to control, compared to vessels treated with TIC10 or FOXO1 inhibitor. These results indicate that treatment with TIC10 exerts vasoprotective effects, however, treatment with a FOXO1 inhibitor slightly worsens relaxation compared to control and when used in combination significantly worsens relaxation of vessels. As described, FOXO1 as well as FOXO3a are linked to increased TRAIL upregulation and further to that increased eNOS activity. Inhibition of FOXO1 therefore prevents TRAIL transcription which is postulated in this study by reduced death receptor expression. Other studies have linked Akt phosphorylation to increased eNOS activity, whereby Akt inhibition stimulates endothelial dysfunction in hypertension.32 TIC10 inhibits Akt and this could explain why FOXO1 inhibition in conjunction with TIC10 treatment worsened relaxation compared to all the other treatment groups, as indicated in figure 1 and the results in the AUC analysis.

FOXO1 inhibition downregulated endothelial expression of DR4 compared to control however not DR5. The FOXO family are known to induce the expression of death receptor ligands fundamental for apoptotic signalling.19 Inhibition of FOXO1 and FOXO3a proteins inhibits transcriptional activity of TRAIL and thus inhibit increased expression of TRAIL ligand death receptors. However, there is no evidence that their inhibition leads to a downregulation of death receptor expression and as such further investigations into the mechanisms associated with FOXO1 inhibition and endothelial DR4 downregulation need to be conducted.

The results of this study show that TIC10 enhances vascular relaxation potentially via a FOXO1/DR5 dependent pathway. It is postulated that TIC10 treatment stimulating the

upregulation of DR5 is a result of elevated TRAIL transcription dependent on FOXO1 activity. Further investigations need to be undertaken to investigate the isolated roles of FOXO1 and FOXO3a factors in response to TIC10 treatment and how they influence death receptor expression and vasodilation. Further investigations into TRAIL also need to be undertaken to investigate the possible relationship between FOXO1 and TRAIL transcription. This study illustrates that TIC10 can target increase vasodilation capacity of blood vessels via mechanisms associated with DR5 upregulation.


All experiments were undertaken in accordance with the “Australian Code of Practise for the Care and Use of Animals for Scientific Purposes,” with all experiments and protocols approved by the Victoria University Animal Ethics Committee.


White New Zealand male Rabbits (12 weeks old, n=6, VUAEC #12/019) were sedated using 0.25mg/kg medetomidine and then anaesthetised using 4% isoflurane. Rabbits were exsanguinated and the abdominal aorta flushed using cold KREBS (118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4·7H2O, 1.2 mmol/L KH2PO4, 25 mmol/L NaHCO3, and 11.7 mmol/L glucose; obtained from Sigma Aldrich, St. Louis, MO, USA). Once excised, vessels were cleaned of fat and connective tissue, cut into 3mm rings and immediately placed into organ baths (OB8, Zultek Engineering, Australia).

Experimental protocol Isometric tension

Vessels were placed into organ baths containing fresh KREBS solution maintained at 37C and bubbled with carbogen, to acclimatise for 30 minutes. Vessels were then placed onto transducer wires and stretch to 2g. After 30 minutes, vessels were stretched to 2g and refreshed with KREBS, after another 30 minutes vessels were re-stretched to 2g. The experiment contained four treatment groups these included a control, TIC10, FOXO1 inhibitor (AS1842856) (5x10-7mM) and a

combinatory FOXO1 inhibitor with TIC10 treatment group. TIC10 (5µM) was incubated for 2 hours. In the FOXO1 inhibitor plus TIC10 treatment group, 15 minutes prior to TIC10 administration, vessels were pre-incubated with AS1842856). After 2-hours, vessels were pre-
constricted using 3×10-7 M phenylephrine. Once a plateau was reached, an accumulative dose response curve to acetylcholine was performed (10-8 – 10-5 M, half log units).


Vessels used in the in-vitro isometric tension studies were immediately removed at the completion of the protocol and fixed in 4% paraformaldehyde for 24 hours, before being transferred into 1xPBS and refrigerated at 4°C. Once tissues were fixed, they were embedded into a paraffin wax block and sliced using a manually operated rotary microtome, CUT 4060 into 5-micron thicknesses and placed onto microscopic slides. Slides were deparaffinised, re-hydrated and placed into 10mM TRIS solution (pH 7.4) for 5 minutes. Then, goat serum (1%, room temperature) was placed on the slide on a level stage and allowed to incubate for 20 minutes. Goat serum was tapped off the slide and immediately 200µL of the primary antibodies DR4 (1:100 dilution Sapphire Bioscience, Cat# LS-C340229) and DR5 (1:100 dilution; Sapphire Bioscience, Cat# LS-B2073) were added. The slide was then placed in a hydration chamber overnight at room temperature. Slides were then washed in TRIS solution for five minutes, and the secondary step (IMPRESS kit, Cat#MP-7452) was added and incubated for one hour. After the incubation period slides were rinsed with fresh TRIS for five minutes. DAB solution was then added (2minutes) and then rinsed in DDH2O. Slides were then counterstained with haematoxylin, blued in Scotts tap water, rinsed in DDH2O, dehydrated, and mounted using DPX. Stained tissues were then placed under a microscope at 400x magnification and digital photographs of the tissue were taken using digital camera (BX53, Olympus, USA). Four images per tissue were taken (top, bottom, left and right sides). Images were then transferred into MCID software (MCID CORE; Interface’s Imaging, Linton, UK) for semi quantitative analysis. MCID software used saturation, intensity and hue to indicate the DAB reaction indicative of protein expression. A ribbon tool was used to trace the endothelium and an average taken amongst the sections used. The tracing generated two values; intensity and proportional area which were used to formulate proportional intensity which was used for data analysis, as established in our laboratory.

Statistical analysis
Data was analysed using either a one or two-way ANOVA (GraphPad Prism), followed by a Sidaks multiple comparisons post hoc test. All data is presented as mean ± SEM and significance taken at p<0.05.

Conflict of interest: Authors declare no conflict of interest.


1. Gutierrez J, Alloubani A, Mari M, Alzaatreh M. Cardiovascular Disease Risk Factors:
Hypertension, Diabetes Mellitus and Obesity among Tabuk Citizens in Saudi Arabia. Open Cardiovasc Med J. 2018;12:41‐49.
2Frostegård J. Immunity, atherosclerosis and cardiovascular disease. BMC Med. 2013;11:117.
3Lnsis A. Atherosclenrosis. Nature. 2000;407:233-241.
4Hermann M, Flammer A, Lüscher TF. Nitric oxide in hypertension. J Clin Hypertens (Greenwich). 2006;8(12 Suppl 4):17‐29.
5Galley HF, Webster NR. Physiology of the endothelium. Br J Anaesth. 2004;93(1):105‐113.
6Naseem KM. The role of nitric oxide in cardiovascular diseases. Mol Aspects Med. 2005;26(1-2):33‐65.
7Premer C, Kanelidis AJ, Hare JM, Schulman IH. Rethinking Endothelial Dysfunction as a Crucial Target in Fighting Heart Failure. Mayo Clin Proc Innov Qual Outcomes. 2019;3(1):1‐13.
8Zhao Y, Vanhoutte PM, Leung SW. Vascular nitric oxide: Beyond eNOS. J Pharmacol Sci. 2015;129(2):83‐94.
9Allen JE, Krigsfeld G, Mayes PA, et al. Dual inactivation of Akt and ERK by TIC10 signals Foxo3a nuclear translocation, TRAIL gene induction, and potent antitumor effects. Sci Transl Med. 2013;5(171):171ra17.

10Allen JE, Krigsfeld G, Patel L, et al. Identification of TRAIL-inducing compounds
highlights small molecule ONC201/TIC10 as a unique anti-cancer agent that activates the TRAIL pathway. Mol Cancer. 2015;14:99.
11Greer YE, Lipkowitz S. TIC10/ONC201: a bend in the road to clinical development. Oncoscience. 2015;2(2):75‐76.
12Prabhu VV, Allen JE, Dicker DT, El-Deiry WS. Small-Molecule ONC201/TIC10 Targets Chemotherapy-Resistant Colorectal Cancer Stem-like Cells in an Akt/Foxo3a/TRAIL- Dependent Manner. Cancer Res. 2015;75(7):1423‐1432.
13Xu J, Zhou JY, Wei WZ, Wu GS. Activation of the Akt survival pathway contributes to TRAIL resistance in cancer cells. PLoS One. 2010;5(4):e10226.
14Watt V, Chamberlain J, Steiner T, Francis S, Crossman D. TRAIL attenuates the development of atherosclerosis in apolipoprotein E deficient mice. Atherosclerosis. 2011;215(2):348‐354.
15Nho RS, Hergert P. FoxO3a and disease progression. World J Biol Chem. 2014;5(3):346‐354.
16Zhang X, Tang N, Hadden TJ, Rishi AK. Akt, FoxO and regulation of apoptosis. Biochim Biophys Acta. 2011;1813(11):1978‐1986.
17Potente M, Urbich C, Sasaki K, et al. Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization. J Clin Invest. 2005;115(9):2382‐2392.
18Oellerich MF, Potente M. FOXOs and sirtuins in vascular growth, maintenance, and aging. Circ Res. 2012;110(9):1238‐1251.
19Fu Z, Tindall DJ. FOXOs, cancer and regulation of apoptosis. Oncogene. 2008;27(16):2312‐2319.
20Kumar R, Herbert PE, Warrens AN. An introduction to death receptors in apoptosis. Int J Surg. 2005;3(4):268‐277.
21Modur V, Nagarajan R, Evers BM, Milbrandt J. FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression. Implications for PTEN mutation in prostate cancer. J Biol Chem. 2002;277(49):47928‐47937.
22Wang Y, Zhou Y, Graves DT. FOXO transcription factors: their clinical significance and regulation. Biomed Res Int. 2014;2014:925350.
23Liu Y, Ao X, Ding W, et al. Critical role of FOXO3a in carcinogenesis. Mol Cancer. 2018;17(1):104.

24Lee HY, Youn SW, Cho HJ, et al. FOXO1 impairs whereas statin protects endothelial
function in diabetes through reciprocal regulation of Kruppel-like factor 2. Cardiovasc Res. 2013;97(1):143‐152.
25Zhou J, Liao W, Yang J, et al. FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway. Autophagy. 2012;8(12):1712‐1723.
26Morris JB, Kenney B, Huynh H, Woodcock EA. Regulation of the proapoptotic factor FOXO1 (FKHR) in cardiomyocytes by growth factors and alpha1-adrenergic agonists. Endocrinology. 2005;146(10):4370‐4376.
27Talekar MK, Allen JE, Dicker DT, El-Deiry WS. ONC201 induces cell death in pediatric non-Hodgkin’s lymphoma cells. Cell Cycle. 2015;14(15):2422‐2428.
28Truneh A, Sharma S, Silverman C, et al. Temperature-sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor. J Biol Chem. 2000;275(30):23319‐23325.
29Chao SC, Chen YJ, Huang KH, et al. Induction of sirtuin-1 signaling by resveratrol induces human chondrosarcoma cell apoptosis and exhibits antitumor activity. Sci Rep. 2017;7(1):3180.
30Xia N, Strand S, Schlufter F, et al. Role of SIRT1 and FOXO factors in eNOS transcriptional activation by resveratrol. Nitric Oxide. 2013;32:29‐35.
31Potente M, Ghaeni L, Baldessari D, et al. SIRT1 controls endothelial angiogenic functions during vascular growth. Genes Dev. 2007;21(20):2644‐2658.
32Iaccarino G, Ciccarelli M, Sorriento D, et al. AKT participates in endothelial dysfunction in hypertension. Circulation. 2004;109(21):2587‐2593.

Figure legends

Figure 1: Dose response graph of treatment groups illustrating % relaxation of cumulative dose response to acetylcholine. Graph shows enhanced vascular relaxation of control vessels treated with TIC10 (Red line) at Ach concentrations 10-7 M and 10-6.5 M. Combination therapy with FOXO1 inhibitor and TIC10 worsened vascular relaxation compared to both Control and TIC10 treated vessels at varying Ach doses.

Figure 2: Br graph representing average area under the curve (AUC)  SEM, describing the drug concentration as a function of time. Significance P<0.05 is observed for Control vs. FOXO1 Inhibitor + TIC10, TIC10 vs. FOXO1 Inhibitor + TIC10 and TIC10 vs. FOXO1 inhibitor treatment groups obtained by One-way ANOVA. Significance was also observed between the Control and TIC10 group, indicated by an unpaired t-test.

Figure 3: Bar graph representing DR5 expression in the endothelium of abdominal aorta rings incubated with TIC10, FOXO1 inhibitor, TIC10 and a FOXO1 inhibitor and control vessels. Endothelial upregulation of DR5 is observed in vessels incubated with TIC10. Addition of

FOXO1 inhibitor has no effect on DR5 expression, however, inhibits TIC10 stimulated DR5 expression.

Figure 4: Images 400x magnification depicting immunohistochemical staining for DR5 in both the absence and presence of a FOXO1 inhibitor. Brown precipitate indicates positive immunolocalization of DR5 expression. (A) Control vessels photos showing limited DR5 expression (B) Vessels pre-incubated with TIC10 showing high levels of endothelial DR5 expression.(C) TIC10+FOXO1 inhibitor DR5 expression. (D) FOXO1 inhibitor showing a clear reduction in endothelial DR5 expression compared to the TIC10 treatment alone.

Figure 5: Bar graph illustrating DR4 downregulation in endothelial cells following pre-incubation with FOXO1 inhibitor compared to control (0.37±0.07 vs. 1±0.19, p<0.05). Although there is a slight increase in endothelial DR4, there is no significant upregulation in response to TIC10 treatment.

Figure 6: Images of DR4 expression in both the absence and presence of the FOXO1 inhibitor in response to and without TIC10 treatment.(A) Control vessels showing presence of DR4 expression (B) Vessels pre-incubated with TIC10 showing slightly elevated levels of endothelial DR4 compared to control vessels however no significance was observed. (C) TIC10+FOXO1 inhibitor DR4 downregulation. (D) FOXO1 inhibitor showing downregulation of endothelial DR4.

Table 1. Table indicating significance of each group at each ach concentration for results obtained in the isometric tension studies shown in Figure 1.
[M] Control vs. TIC10 Control vs. TIC10+FOXO1 inhibitor Control vs. FOXO1 inhibitor TIC10 vs. TIC10+ FOXO1 inhibitor TIC10 vs. FOXO1 inhibitor TIC10+ FOXO1 inhibitor vs. FOXO1 inhibitor
10-8 No No No No No
10-7.5 No No No Yes (p=0.0320) No No
10-7 Yes (p=0.0493) Yes (p=0.0345) Yes (p=0.0309) Yes (p=0.008) Yes (p=0.0024) No
10-6.5 Yes (p=0.0209) No No Yes (p=0.0187) Yes (p=0.0493) No
10-6 No No No Yes (p=0.0455) No No
10-5.5 No Yes (p=0.0382) No Yes (p=0.0382) No No
10-5 No Yes (p=0.0413) Yes (p=0.0424) No Yes (p=0.0343) No