title: Vascular effects of anti-cancer drugs
Creators: name: Cameron, Alan C.
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: Anti-cancer drugs, cisplatin, Vascular endothelial growth factor inhibitors (VEGFi), endothelial function.

description: Introduction Anti-cancer therapies have improved the prognosis of patients with cancer to the extent that cancer survival has doubled over recent decades. Improved cancer survival is partly offset by a burden of cardiovascular disease (CVD) associated with anti-cancer therapies. Introduction of drug regimens containing the platinum-derived agent cisplatin for testicular cancer in the 1970s has allowed almost all patients to be cured, although up to 9% of patients treated with cisplatin develop arterial or venous thrombosis. Vascular endothelial growth factor inhibitor (VEGFi) anti-cancer drugs improve outcomes for patients with a range of cancers, although hypertension occurs up to 80% of patients treated with VEGFi. The mechanisms responsible for CVD associated with cisplatin and VEGFi remain undefined, although effects on the endothelium appear to be of fundamental importance. Cisplatin exerts endothelial effects that may contribute to endothelial dysfunction and accelerated CVD, although the time-course of effects from cisplatin on the endothelium and risk of CVD are not fully understood. Hypertension associated with VEGFi may relate to changes in endothelial vasomotor tone through upregulation of the potent vasoconstrictor endothelin-1 (ET-1) and reduced bioavailability of the vasodilator nitric oxide (NO). Given that the mechanisms underlying CVD associated with cisplatin and VEGFi are incompletely understood, current strategies for prevention and management are generic and not pathophysiologically targeted. I hypothesised that cisplatin and VEGFi anti-cancer drugs exert differential endothelial effects that are responsible for associated cardiovascular toxicity. I therefore aimed to 1) evaluate temporal changes in vascular function and cardiovascular biomarkers in cisplatin-treated patients with testicular cancer; 2) evaluate the endothelial vasomotor and fibrinolytic effects of VEGFi in healthy males using forearm arterial plethysmography, in the presence and absence of endothelin receptor (ETR) antagonism; and 3) investigate the effects of cisplatin and VEGFi on downstream signalling pathways associated with hypertension and thrombosis in human aortic endothelial cells (HAECs). Early Vascular Effects of Cisplatin I conducted a prospective evaluation of changes in endothelial function and cardiovascular biomarkers in 27 patients with testicular cancer attending the Beatson West of Scotland Cancer Centre between January 2016 and July 2017. Patients were stratified into 3 groups according to management: 1) active surveillance [orchidectomy alone] (n=10); 2) orchidectomy plus 1-2 cycles of adjuvant cisplatin-based chemotherapy (n=7); or 3) orchidectomy plus 3-4 cycles of cisplatin-based chemotherapy for metastatic disease (n=10). Assessment of endothelial vasomotor function was made using the Angiodefender system which provides a measure of percentage forearm arterial percentage flow-mediated dilatation (% FMD). Venous blood was collected for subsequent assessment of vascular biomarkers. Participants attended for 6 visits over 9 months: the initial assessment was within 8 weeks of orchidectomy and subsequent assessments were within 24 hours of the initial cisplatin regimen and at 6 weeks, 3 months, 6 months and 9 months. In patients managed with the metastatic disease cisplatin regimen, % FMD acutely decreased within 24 hours of the first cisplatin dose and returned to baseline thereafter. Serum cholesterol and glycated haemoglobin (HbA1C) increased at 6 weeks in patients managed with the metastatic disease cisplatin regimen and were unchanged from baseline at 9 months. My findings demonstrate that cisplatin-based chemotherapy induces acute and transient endothelial dysfunction, hypercholesterolaemia and hyperglycaemia in the early period following treatment, and highlight therapeutic targets to reduce associated CVD that are mechanistically relevant and appropriately timed. Later Vascular Effects of Cisplatin I conducted a case-control study of 26 patients with testicular cancer managed 1 to 7 years previously. Patients were stratified into 2 groups by management: 1) orchidectomy plus cisplatin-based chemotherapy (n=12); or 2) orchidectomy alone (n=14). Forearm blood flow (FBF) responses to intra-arterial bradykinin (BK; 100, 300 and 1000 pmol/minute), acetylcholine (ACh; 5, 10 and 20 µg/minute), and sodium nitroprusside (SNP; 2, 4 and 8 µg/minute) were measured using forearm venous occlusion plethysmography. Venous blood sampling was performed to assess local release of the fibrinolytic factor tissue plasminogen activator (tPA) and its main inhibitor, plasminogen activator inhibitor-1 (PAI-1). The primary analysis compared responses in all patients managed with cisplatin versus surveillance and a secondary analysis compared responses in patients 12-18 months versus 5-7 years after treatment. Intra-arterial BK, ACh and SNP evoked dose-dependent vasodilation in all participants. Vasodilator and fibrinolytic factor responses were not different in patients managed with cisplatin-based chemotherapy compared to active surveillance and there were no differences when assessment was made in patients 12-18 months or 5-7 years after treatment. The results demonstrate that cisplatin-based chemotherapy is not associated with impaired endothelial function in the medium-later period following treatment. Taken together with results of my study assessing early effects of cisplatin, this suggests that adverse vascular effects of cisplatin are predominantly confined to the early period surrounding treatment and strategies to prevent associated CVD should be focused on this period. Endothelial Effects of VEGFi I performed venous occlusion plethysmography studies to explore effects of intra-arterial bevacizumab (a VEGFi) on FBF, ET-1 concentrations and fibrinolytic factor release in the presence and absence of ETAR antagonism (BQ-123). 38 healthy male participants were recruited to the study. FBF responses and ET-1 concentrations were assessed for up to 2 hours following intra-arterial infusion of bevacizumab 36, 72 and 144 µg/dL forearm volume/minute for 15 minutes [n = 6, 6 and 8, respectively] and 36 µg/dL for 60 minutes [n=8]. Resting FBF and local ET-1 concentrations were not significantly altered by bevacizumab for up to 2 hours following any infusion. In a separate cohort, 10 participants attended for 2 visits separated by at least 2 weeks. Intra-arterial BK 100 and 1000 pmol/minute was infused in the absence and presence of bevacizumab 144 µg/dL forearm volume/minute, alongside intra-arterial BQ-123 (10 nmol/minute) throughout one visit and placebo during the other. BK dose-dependently increased vasodilatation and tPA release, responses that were not significantly altered by bevacizumab or BQ-123. My results demonstrate that bevacizumab does not acutely alter resting vascular tone or BK-induced vasorelaxation and fibrinolytic factor release in healthy young males, although this may differ in cancer patients treated chronically with VEGFi and warrants further investigation. Effects from Cisplatin and VEGFi on Human Aortic Endothelial Cells I made an assessment of the in vitro effects of cisplatin and VEGFi upon markers of vasoconstriction, vasodilation, thrombosis, inflammation and stress kinase signalling. I stimulated HAECs with cisplatin (1, 3 or 15 g/ml) for 5 minutes, 15 minutes and 24 hours; and vascular endothelial growth factor (VEGF, 10 ng/ml), vatalanib (VEGFi, 100 nmol/), VEGF (10 ng/ml) and vatalanib (100 nmol/L) in combination for 5 and 15 minutes. I assessed effects on endothelial nitric oxide synthase (eNOS) phosphorylation and stress kinase pathways [p38 mitogen-activated protein kinase (p38 MAPK), extra-cellular signal-regulated kinases 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK) and protein kinase B (Akt)] by Western blot analysis. I performed real-time polymerase chain reaction (PCR) to probe mediators of vasoconstriction (pre-pro-ET-1), inflammation [vascular cellular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α), monocyte chemoattractant protein 1 (MCP-1)] and thrombosis [von Willebrand factor (vWF), tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), tissue factor (TF), prostacyclin (PGI2) and platelet activating factor receptor (PAFR)]. Cisplatin decreased messenger ribonucleic acid (mRNA) expression of tPA and increased phosphorylation of Akt, ERK 1/2 and JNK. Vatalanib induced a trend to reduce eNOS phosphorylation and did not alter pre-pro-ET-1 mRNA expression. My findings suggest cisplatin and VEGFi induce differential effects on downstream signalling pathways at the level of HAECs. Cisplatin reduces tPA mRNA expression and activates stress kinase signalling pathways, processes that may occur as a result of direct endothelial toxicity. Notwithstanding limitations due to small sample size, VEGFi tends to reduce eNOS phosphorylation and does not directly affect ET-1 signalling. The data lend support to the hypothesis that thrombotic CVD associated with cisplatin is due to endothelial toxicity resulting in a pro-thrombotic state, and suggest that decreased NO activity may be an important early factor contributing to changes in vascular tone underpinning VEGFi-associated hypertension. Summary I explored mechanisms underlying CVD associated with cisplatin and VEGFi anti-cancer drugs in studies performed in cancer patients, healthy volunteers and using in vitro approaches. I have demonstrated that cisplatin induces acute and transient endothelial dysfunction, hypercholesterolaemia and hyperglycaemia in patients. It reduces the production of fibrinolytic factors and increases stress kinase signalling in cell culture. In healthy volunteers, I demonstrated that acute exposure to VEGFi does not affect resting vasomotor tone, ET-1 concentrations or BK-induced vasodilatation and fibrinolytic factor release. In HAECs, VEGFi induces a trend to reduced eNOS phosphorylation. My results highlight mechanistically relevant and time-appropriate strategies for approaches to prevent and treat CVD associated with cisplatin and VEGFi. A trial of focused, short-term cardiovascular-protective therapy should be considered in patients treated with cisplatin. The role of ET-1 and NO in the development of VEGFi-associated hypertension should be further examined in patients receiving VEGFi over a more prolonged period. These findings provide additional evidence that mechanisms of drug-induced vascular toxicity are diverse. Preventative and therapeutic strategies need to include careful consideration of pathophysiological mechanisms. Only by doing so will the optimum balance between cardiovascular protection and successful anti-cancer therapy be achieved.