The vascular endothelial growth factor trap aflibercept induces vascular dysfunction and hypertension via attenuation of eNOS/NO signaling in mice

Abstract

Aflibercept, as a soluble decoy vascular endothelial growth factor receptor, Which has been used as a first-line monotherapy for cancers. Aflibercept often causes cardiovascular toxicities including hypertension, but the mechanisms underlying aflibercept-induced hypertension remain unknown. In this study we investigated the effect of short-term and long-term administration of aflibercept on blood pressure (BP), vascular function, NO bioavailability, oxidative stress and endothelin 1 (ET-1) in mice and cultured endothelial cells. We showed that injection of a single-dose of aflibercept (18.2, 36.4 mg/kg, iv) rapidly and dose-dependently elevated BP in mice. Aflibercept treatment markedly impaired endothelial-dependent relaxation (EDR) and resulted in NADPH oxidases 1 (NOX1)- and NADPH oxidases 4 (NOX4)-mediated generation of ROS, decreased the activation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) concurrently with a reduction in nitric oxide (NO) production and elevation of ET-1 levels in mouse aortas; these effects were greatly attenuated by supplementation of L-arginine (L-arg, 0.5 or 1.0 g/kg, bid, ig) before aflibercept injection. Similar results were observed in L-arg-pretreated cultured endothelial cells, showing markedly decreased ROS accumulation and AKT/eNOS/NO signaling impairment induced by aflibercept. In order to assess the effects of long-term aflibercept on hypertension and to evaluate the beneficial effects of L-arg supplementation, we administered these two drugs to WT mice for up to 14 days (at an interval of two days). Long-term administration of aflibercept resulted in a sustained increase in BP and a severely impaired EDR, which are associated with NOX1/NOX4-mediated production of ROS, increase in ET-1, inhibition of AKT/eNOS/NO signaling and a decreased expression of cationic amino acid transporter (CAT-1). The effects caused by long-term administration were greatly attenuated by L-arg supplementation in a dose-dependent manner. We conclude that aflibercept leads to vascular dysfunction and hypertension by inhibiting CAT-1/AKT/eNOS/NO signaling, increasing ET-1, and activating NOX1/NOX4-mediated oxidative stress, which can be suppressed by supplementation of L-arg. Therefore, L-arg could be a potential therapeutic agent for aflibercept-induced hypertension.

Keywords: L-arginine; cultured endothelial cells; aflibercept; endothelial dysfunction; hypertension; superoxide production; endothelin 1; cationic amino acid transporter-1.

Fig. 1. A single dose of aflibercept leads to an acute increase in SBP and impairment of vascular function.

a SBP was measured by the noninvasive tail-cuff method before and after treatment with a single dose of saline or aflibercept (n = 8; *P < 0.05 versus the saline group; ^#P < 0.05 versus the low-dose aflibercept group). b–d Representative SBP recordings from WT mice 3 days before aflibercept treatment as a baseline (b) and 1–4 days after infusion of a single dose of aflibercept at 18.4 mg/kg (c) or 36.4 mg/kg (d) using radiotelemetric BP measurements (n = 6). e, f Dose-response curves for endothelium-dependent (e) and endothelium-independent relaxation (f) at different times (1–4 days) after treatment with 36.4 mg/kg aflibercept (n = 8; *P < 0.05 versus the saline group; ^#P < 0.05 versus the 4th day after aflibercept treatment group). g Representative images of H&E staining of thoracic aortas. Wall thickness in each group (n = 6). The data are presented as the mean ± SEM; n represents the number of samples or mice; NS no significant difference; SBP systolic blood pressure; ACh acetylcholine; SNP sodium nitroprusside.

Fig. 2. A single dose of aflibercept results in NOX1/NOX4-mediated ROS accumulation and impairs AKT/eNOS/NO signaling.

a Representative images of DHE staining of aortic segments after injection of a single dose of 36.4 mg/kg aflibercept, and DHE fluorescence intensity from the data representing ROS levels on days 1–4 after aflibercept injection (n = 5; scale bar; 50 μm; *P < 0.05 versus the saline group). b Nitrotyrosine was assessed as an index of peroxynitrite (ONOO⁻) formation in aorta (n = 7–8; *P < 0.05 versus saline group). c Immunoblotting analysis of NOX1 and NOX4 protein expression in the aorta before and after injection of 36.4 mg/kg aflibercept and protein abundance from the experiments was shown (n = 4; *P < 0.05 versus the saline group; ^#P < 0.05 versus the 3rd day after aflibercept treatment group). d, e Aflibercept significantly reduced NO levels in serum and aortic tissues (n = 6 or 7; *P < 0.05 versus the saline group; ^#P < 0.05 versus the 4th day after aflibercept treatment group). f Aflibercept significantly increased ET-1 levels in serum (n = 6 or 7; *P < 0.05 versus the saline group). g Representative immunoblots showing VEGFR1 and VEGFR2 protein expression in the aorta; Protein expression levels of VEGFR1 and VEGFR2 before and after aflibercept injection (n = 4). h Representative immunoblots showing p-AKT (Ser473), AKT, p-eNOS (Ser1177), and eNOS expression in the aorta, and quantification of protein levels from the data were shown (n = 4; *P < 0.05 versus the saline group; ^#P < 0.05 versus the 3rd day after aflibercept treatment group). The data are presented as the mean ± SEM; n represents number of samples or mice; NS no significant difference; Akt protein kinase B; p-Akt phosphorylated Akt; eNOS endothelial nitric oxide synthase; p-eNOS phosphorylated eNOS.

Fig. 3. L-arg supplementation prevents hypertension, impairment of EDR, ROS accumulation, and reduction in NO production induced by a single dose of aflibercept.

a SBP was measured by the noninvasive tail-cuff method in mice treated with saline or L-arg (0.5 or 1.0 g· kg⁻¹ ·d⁻¹) before and after injection of a single dose of 36.4 mg/kg aflibercept (n = 6; *P < 0.05 versus the saline group; ^#P < 0.05 versus the low-dose L-arg group). b Following injection of a single dose of aflibercept, NO levels in the aortic tissues were measured using a nitric oxide assay kit in the presence or absence of L-arg (1.0 g· kg⁻¹· d⁻¹) on days 1 and 3 (n = 6; *P < 0.05 versus the saline group; ^#P < 0.05 versus the L-arg group; ^&P < 0.05 versus the 1st day after aflibercept + L-arg treatment group; ^$P < 0.05 versus the 3rd day after aflibercept + L-arg treatment group). c, d Dose-response curves for endothelium-dependent (c) and endothelium-independent relaxation (d) were constructed on days 1 and 3 after 36.4 mg/kg aflibercept injection in the presence or absence of L-arg (1.0 g· kg⁻¹ ·d⁻¹) (n = 7 or 8; *P < 0.05 versus the saline group). e, f Representative images of DHE staining of aortic segments (e) and DHE fluorescence intensity data demonstrating that aflibercept-induced ROS accumulation was reversed by L-arg (f) (n = 5; *P < 0.05 versus the saline group; ^#P < 0.05 versus the L-arg group; ^&P < 0.05 versus the 1st day after aflibercept + L-arg treatment group; ^$P < 0.05 versus the 3rd day after aflibercept + L-arg treatment group). g ET-1 levels in aortic tissues were measured after administration of a single dose of aflibercept on days 1 and 3 in the absence or presence of L-arg (n = 6; *P < 0.05 versus the saline group; ^&P < 0.05 versus the 1st day after aflibercept + L-arg treatment group; ^$P < 0.05 versus the 3rd day after aflibercept + L-arg treatment group). The data are presented as the mean ± SEM; n represents the number of samples or mice; NS no significant difference.

Fig. 4. L-arg prevents against aflibercept-induced ROS accumulation and impairment of AKT/eNOS/NO in cultured endothelial cells.

a Green fluorescence observed by confocal microscopy in cells preloaded with DAF-FM representing intracellular NO levels, and integrative fluorescence per image demonstrating that the aflibercept-induced reduction in NO levels was rescued by L-arg (n = 6; *P < 0.05 versus the saline group; ^#P < 0.05 versus the aflibercept group). b Representative DHE staining of HUVECs and DHE fluorescence intensity data demonstrating that aflibercept-induced ROS accumulation was reserved by L-arg in each group (n = 6; *P < 0.05 versus the saline group; ^#P < 0.05 versus the aflibercept group). c–f Representative immunoblots showing NOX1, NOX4, p-AKT (Ser473), AKT, p-eNOS (Ser1177), eNOS, and CAT-1 expression in HUVECs (c) and quantification of protein expression levels from the data shown in (d–f) (n = 4; *P < 0.05 versus the saline group; ^&P < 0.05 versus the L-arg group; ^#P < 0.05 versus the aflibercept group). g The intracellular L-arg concentration in HUVECs was measured by the L-Arginine Assay Kit, and the data showed that the aflibercept-induced decrease in intracellular L-arg was greatly attenuated by extracellular L-arg supplementation (n = 6; *P < 0.05 versus the saline group; ^&P < 0.05 versus the L-arg group; ^#P < 0.05 versus the aflibercept group). The data are presented as the mean ± SEM; n represents the number of samples or mice.

Fig. 5. Long-term application of aflibercept-induced hypertension and vascular dysfunction in mice.

a The SBP of each group was measured by the noninvasive tail-cuff method for up to 14 days; the data demonstrated that L-arg supplementation prevented the sustained increase in SBP induced by long-term aflibercept injection (n = 7 or 8; *P < 0.05 versus the saline group; ^#P < 0.05 versus the aflibercept + low-dose L-arg group; ^&P < 0.05 versus the Ang II group). b–g Representative SBP recordings from radiotelemetric BP measurements from WT mice 3 days before aflibercept treatment as a baseline (b) and from those administered L-arg, aflibercept and L-arg + aflibercept showing the raw data generated on the last 3 days (c–f), with Ang II (490 ng ·kg⁻¹ ·min⁻¹) (g) as a positive control (n = 6). h, i Dose-response curves for endothelium-dependent (h) and endothelium-independent relaxation (i) were constructed on day 14 after injection of 36.4 mg/kg aflibercept in the presence or absence of 0.5 or 1.0 g/kg L-arg (n = 7 or 8; *P < 0.05 versus the saline group; ^#P < 0.05 versus the aflibercept + low-dose L-arg group). The data are presented as the mean ± SEM; n represents the number of samples or mice.

Fig. 6. L-arg ameliorates hypertension and vascular dysfunction induced by long-term application of aflibercept in mice.

a The SBP of each group was measured by the noninvasive tail-cuff method for up to 14 days; consistently, long-term high-dose application of aflibercept led to a sustained increase in SBP, and this dramatic aflibercept-induced increase in SBP was greatly inhibited by L-arg supplementation on day 7 (as indicated by the green arrowhead) (n = 7; *P < 0.05 versus the saline group; ^#P < 0.05 versus the low-dose aflibercept group). b, c Dose-response curves for endothelium-dependent (b) and endothelium-independent relaxation (c) were constructed on day 14 after injection of 36.4 mg/kg aflibercept in the presence of absence of 1.0 g/kg L-arg on 8–14 days (n = 7; *P < 0.05 versus the saline group; ^#P < 0.05 versus the aflibercept group; ^&P < 0.05 versus the low-dose aflibercept group). The data are presented as the mean ± SEM; n represents the number of samples or mice.

Fig. 7. L-arg prevents oxidative stress and the reduction in eNOS/NO signaling induced by long-term application of aflibercept.

a Representative images of DHE staining of aortic segments after injection of a single dose of 36.4 mg/kg aflibercept in the presence or absence of L-arg and the fluorescence intensity data demonstrating that aflibercept-induced ROS accumulation was greatly attenuated by L-arg (n = 6; *P < 0.05 versus the saline group; ^#P < 0.05 versus the aflibercept + low-dose L-arg group; ^&P < 0.05 versus the aflibercept + high-dose L-arg group). b Representative immunoblots showing p-eNOS (Ser1177) and eNOS expression in aortic tissues and quantification of protein abundance demonstrating that the aflibercept-induced decrease in p-eNOS was ameliorated by L-arg (n = 4; *P < 0.05 versus the saline group; ^$P < 0.05 versus the L-arg group; ^#P < 0.05 versus the aflibercept + low-dose L-arg group; ^&P < 0.05 versus the aflibercept + high-dose L-arg group). c NO levels in aortic tissues were measured by the Nitric Oxide Assay Kit on day 14 in the absence or presence of L-arg following aflibercept injection (n = 6; *P < 0.05 versus the saline group; ^$P < 0.05 versus the L-arg group; ^#P < 0.05 versus the aflibercept + low-dose L-arg group; ^&P < 0.05 versus the aflibercept + high-dose L-arg group). d–f Representative images of H&E staining of thoracic aortas (d); Wall thickness data (e) and the percentage of the fibrotic area in each indicated group (n = 6) (f). The data are presented as the mean ± SEM; n represents number of samples or mice; NS no significant difference.