The Activation of Endothelial Cells Relies on a Ferroptosis-Like Mechanism: Novel Perspectives in Management of Angiogenesis and Cancer Therapy

Abstract

The activation of endothelial cells (ECs) is a crucial step on the road map of tumor angiogenesis and expanding evidence indicates that a pro-oxidant tumor microenvironment, conditioned by cancer metabolic rewiring, is a relevant controller of this process. Herein, we investigated the contribution of oxidative stress-induced ferroptosis to ECs activation. Moreover, we also addressed the anti-angiogenic effect of Propranolol. We observed that a ferroptosis-like mechanism, induced by xCT inhibition with Erastin, at a non-lethal level, promoted features of ECs activation, such as proliferation, migration and vessel-like structures formation, concomitantly with the depletion of reduced glutathione (GSH) and increased levels of oxidative stress and lipid peroxides. Additionally, this ferroptosis-like mechanism promoted vascular endothelial cadherin (VE-cadherin) junctional gaps and potentiated cancer cell adhesion to ECs and transendothelial migration. Propranolol was able to revert Erastin-dependent activation of ECs and increased levels of hydrogen sulfide (H₂S) underlie the mechanism of action of Propranolol. Furthermore, we tested a dual-effect therapy by promoting ECs stability with Propranolol and boosting oxidative stress to induce cancer cell death with a nanoformulation comprising selenium-containing chrysin (SeChry) encapsulated in a fourth generation polyurea dendrimer (SeChry@PURE_G4). Our data showed that novel developments in cancer treatment may rely on multi-targeting strategies focusing on nanoformulations for a safer induction of cancer cell death, taking advantage of tumor vasculature stabilization.

Keywords: angiogenesis; endothelial cell hyperactivation; ferroptosis; lipid peroxidation; oxidative stress; polyurea dendrimers; propranolol; tumor vasculature stabilizers.

Figure 1

Erastin (Era) promotes increased levels of ROS-induced lipid peroxides and Propranolol (Prop), through the generation of hydrogen sulfide (H₂S), is able to revert it. (A) The levels of intracellular ROS (DCF-DA) decrease upon Prop exposure, and Era, although increasing the ROS levels, when co-administrated with Prop the levels are similar to the control, at 6 and 16h. (B) The levels of mitochondrial ROS, assessed by MitoSox, are not affected by the presence of Era and/or Prop, at 6 and 16h. (C) Era induces lipid peroxides (C11-Bodipy) generation and although Prop alone did not affect the lipid peroxides content, its combination with Era reverts the levels generated by Era, being this effect more prominent at 16h. (D) The levels of GSH (total and LT:free total) decrease upon exposure to Era and/or Prop for 16h. (E) The variation of the extracellular levels of cysteine (Cys) indicate that Era inhibits the uptake of Cys by HUVECs, while Prop does not interfere with this process. (F–H) Show the regulation of transcriptional expression of genes encoding, respectively, prostaglandin-endoperoxide synthase 2 (PTGS2), glutathione peroxidase 4 (GPX4) and glutathione synthetase (GSS). Erastin decreases significantly GPX4 and PTGS2 expression and tend to decrease GSS expression, being this effect rescued by propranolol. (I) Era does not affect HUVECs death (annexin V plus PI positive cells) while 16h of Prop exposure, with and without Era, increases the ratio of HUVECs death. (J) Era does not affect H₂S levels of HUVECs while Prop increases, at 16h. In graphs the dashed line represents the control condition. All data are normalized to the control condition and represented as mean ± SD. *p<0.5, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2

The ferroptosis-like mechanism driven by Erastin (Era) promotes endothelial cell (ECs) activation and Propranolol (Prop) impairs the phenotype induced by Era exposure. (A) The ferroptosis-like mechanism, generated by Era exposure, promotes HUVECs proliferation (increased ratio of Ki67⁺ (green) nuclei/total nuclei), while Prop decreases the rate of HUVECs proliferation and impairs the phenotype induced by Era. The panel shows representative microscope images of the Ki67 staining. (B) Era fosters HUVECs migration (increased % wound closure) and Prop inhibits and reverts the phenotype induced by Era. (C, D) Era increases the branch point density of vessel-like structures (proxy for vascular density) at the same range of H₂O₂ (ROS; positive control), with no additive effect. Prop, besides the impairment of vessel-like structures formation (decreased branch point density), inhibits the phenotype induced by Era, even when Prop is added to the vessel-like structures already formed in the presence of Era (Era+Prop (2h)) and contrariwise (Prop+Era (2h)). In graphs the dashed line represents the control condition. All data are normalized to the control condition and represented as mean ± SD. **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3

Erastin (Era) promotes the generation of a leakier EC monolayer while increases cancer cell-EC interaction and transendothelial migration. (A) The ferroptosis-like mechanism driven by Era promotes an increased generation of intercellular VE-Cadherin (VE-Cad) gaps per 100µm², while Propranolol (Prop) is able to revert this phenotype. The panel shows representative images (scale: 10 µm) of VE-Cadherin (green) intercellular junctional gaps (arrows) in HUVECs exposed to Era and/or Prop for 16h. (B) Quantification of VE-Cad junctional gaps. (C) Immunofluorescence for ICAM and VCAM detection. (D) ICAM intensity per cell (HUVECs; A.U.: arbitrary units) increases upon Era exposure and although Prop alone does not affect ICAM expression, it is able to abrogate the expression induced by Era, at 16h. (E) VCAM intensity per cell (A.U.: arbitrary units) shows a tendency to increase under Era exposure. The ferroptosis-like mechanism, induced by Era, promotes cancer cell (MDA-MB-231-calcein labelled cells) adhesion to HUVECs (F) and transendothelial migration (G). Prop alone has no effect but impairs cancer cell adhesion and transendothelial migration induced by Era. The panels show representative microscope images (scale: 100 µm; MDA-MB-231-calcein labelled cells (green)). All data are normalized to the control condition and represented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001.

Figure 4

Erastin (Era) and Propranolol (Prop) do not affect the differentiation route of monocytes into ECs. (A) Neither Era and/or Prop affect the expression of vWF, even when co-exposed with a short H₂O₂ stimulation (positive control of the differentiation pattern of monocytes), indicating that Era and/or Prop have no impact in the differentiation process of monocytes-derived cells into ECs. Era and/or Prop exposure before the short H₂O₂ stimulation does not influence the intracellular ROS (DCF-DA; B) and lipid peroxide (11C-Bodipy; C) levels. In (B, C) data are normalized to the control condition and represented as mean ± SD. *p<0.5, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5

SeChry@PUREG₄ plus Propranolol (Prop) increases cancer cell death through the generation of oxidative stress, while in ECs Prop acts as an antioxidant, reverting ROS levels induced by SeChry@PUREG₄. (A) SeChry@PUREG₄ (160 µM and 200 µM) exposure promotes cancer cell death (MDA-MB-231), being this effect boosted by Prop. (B) ECs (HUVECs) are more resistant to SeChry@PUREG₄ -induced cell death, even under Prop exposure. (C–F) Contrarily to HUVECs, in MDA-MB-231 Prop alone increases (C, D) intracellular ROS (DCF-DA) and (E, F) lipid peroxide (11C-Bodipy) levels and it does not revert the generation of ROS-induced lipid peroxidation induced by SeChry@PUREG₄. (G) SeChry@PUREG₄ does not impact the generation of VE-Cad intercellular junctional gaps. The panel shows representative images (scale: 10 µm) of VE-Cadherin (green) intercellular junctional gaps (arrows). In graphs the dashed line represents the control condition. All data are normalized to the control condition and represented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6

Taking advantage of the differential oxidative stress response of cancer cells and endothelial cells (ECs). (A) A pro-angiogenic ferroptosis-like mechanism, through the generation of ROS, accumulation of lipid peroxides and glutathione (GSH) depletion, is implicated in the promotion of ECs hyperactivation, vessels leakiness and cancer cell adhesion and intravasation. (B) Propranolol (Prop) ROS scavenging activity is anti-angiogenic, impairing ECs activation underlined by the ferroptosis-like mechanism. (C) The combination of SeChry@PURE_G4 nanoparticles and Prop was unraveled as a potential cancer therapy. SeChry@PURE_G4 induces cancer cell death mediated by pro-oxidative features, while Prop stabilizes ECs and prevents the formation of a leakier vasculature, avoiding metastasis. Prop enhances the pro-oxidative features of Sechry@PURE_G4 effect.