N-Acetylcysteine Amide Alleviates Cyclophosphamide-Induced Endothelial Damage
Authors: Hassan Moustapha , Ljungman Per , He Rui , Norgren Svante , Zheng Wenyi , Zhao Ying , Månsson-Broberg Agneta , Wardell Eva , Slof Terra 
Published: November 2021

Abstract
Rui He 1,2, Wenyi Zheng 1,2, Terra Slof 3, Eva Wärdell 4, Agneta Månsson-Broberg 4, Svante Norgren 5, Per Ljungman 2, Ying Zhao 1, 2 , Moustapha Hassan 1, 2
Experimental Cancer Medicine, Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, 141 86 Huddinge, Sweden Center of Allogeneic Stem Cell Transplantation (CAST) and Clinical Research Center, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden. Institute for Life Sciences and Chemistry, HU University of Applied Sciences Utrecht, Oudenoord 330, 3513 EX Utrecht, Netherlands Department of Medicine, Division of Cardiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. Children´s and Women´s Health Theme, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden

Aim
The current study aimed to investigate the protective role of the novel antioxidant N-acetylcysteine amide (NACA) in cyclophosphamide (Cy)-induced endothelial injury and to explore the underlying mechanism.
Background
The alkylating agent Cy is still one of the important corner stones in cancer treatment. Cy is used also as a part of the conditioning regimens prior to hematopoietic cell transplantation (HCT) and as a prophylactic treatment post transplantation in graft-versus-host disease (GVHD). Several studies have shown that high doses of Cy are associated with a number of adverse effects including alopecia, gonadal toxicity, hemorrhagic cystitis, carcinogenesis and late cardiovascular toxicity. Moreover, we have shown that treatment with high-dose Cy caused damage to the arterial endothelium, which might contribute to late cardiovascular disorders. Hitherto, there is no prophylactic strategy for Cy-induced endothelial toxicity. Oxidative stress is one of the main players in the pathogenesis of endothelial disorders and hence is an exploitable trait for targeted treatment.
Methods
The prophylactic efficacy of NACA was evaluated using human aortic endothelial cells (HAECs) and human umbilical endothelial cells (HUVECs). The efficacy of NACA was compared with the clinically approved derivative N-acetylcysteine (NAC). Cy is a prodrug that cannot be activated in the endothelial cells. Therefore, cells were incubated with Cy active metabolite, 4-hydroxycyclophosphamide (4-OH-Cy). In order to reveal the underling mechanism of action, we have investigated and evaluated the following parameters: alteration in cell morphology, cell death pathway, inhibition of DNA damage, variation of redox hemostasis, and neovascularization.
Results
We found that pre-incubation with 5 mM NACA preserved the normal endothelial cell morphology. However, for the quantification of the obtained results, cell colonies were visualized using crystal violet staining while cell viability was determined using WST-1 assay. Moreover, NACA was found to decrease 4-OH-Cy-induced DNA damage and cell death via caspase-dependent apoptosis in both HAECs and HUVECs. In addition, NACA significantly inhibited 4-OH-Cy-induced oxidative stress by reducing the intracellular levels of reactive oxygen species (ROS) and by improving the cellular antioxidative capacity. Increased activities of catalase and superoxide dismutase (SOD) together with increased levels of reduced glutathione (GSH) were observed. Meanwhile, NACA pre-treatment rebalanced endothelial nitric oxide synthase (eNOS) and arginase I in HAECs, suggesting that NACA may be used as prophylactic strategy to avoid the impaired endothelium-dependent vasodilation caused by 4-OH-Cy. Moreover, data from tube formation assay indicated that NACA preserved the angiogenic capability of endothelial cells that was compromised by 4-OH-Cy through blockage of Notch signaling pathway. Our results showed that in contrast to NAC, its amide derivative NACA showed remarkably superior ability in alleviating the endothelial damage caused by 4-OH-Cy.
Conclusions
In summary, the present investigation showed that the novel antioxidant NACA could be applied to protect against 4-OH-Cy-induced endothelial cell dysfunction and death. Higher bioavailability and better cellular uptake make NACA to a better prophylactic agent compared to NAC. To translate these findings into the clinic, additional in vivo studies are warranted to confirm the protective potential of NACA.

Disclosures
Ljungman: Takeda: Consultancy, Other: Endpoint committee, speaker; Janssen: Other: Investigator; OctaPharma: Other: DSMB; Enanta: Other: DSMB; Merck: Other: Investigator, speaker; AiCuris: Consultancy.