Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Liver growth factor treatment reverses vascular and plasmatic oxidative stress in spontaneously hypertensive rats Luis Condezo-Hoyos a , Silvia M. Arribas a , Fa ´ tima Abderrahim a , Beatriz Somoza b , Marta Gil-Ortega b , Juan J. Dı´az-Gil c , M. Victoria Conde a , Cristina Susin a , and M. Carmen Gonza ´lez a Background: Liver growth factor (LGF) is an albumin– bilirubin complex with antioxidant actions in vitro. In spontaneously hypertensive rats (SHRs), short LGF treatment exerts antihypertensive and antifibrotic effects. Method: We aimed to determine if LGF treatment (4 i.p. injections, 4.5 mg/rat over 12 days) reduces oxidative stress in SHRs using Wistar–Kyoto (WKY) as control strain. We assessed the following: plasma oxidative stress biomarkers [protein-bound malondialdehyde (MDA); protein carbonyls and advanced glycation end products (AGEs)]; superoxide anion basal production in carotid artery-derived vascular smooth muscle cells (VSMCs) detected by dihydroethidium and confocal microscopy; and expression (western blot) and activities (spectroscopic methods) of NADPH and xanthine oxidases, CuZn, Mn and extracellular superoxide dismutases (SODs) and catalase in carotid arteries. Results: LGF treatment had the following effects: reversed the increase in plasma MDA and protein carbonyls and VSMC superoxide anion levels observed in SHRs, without any effect on WKY strain; reversed the alterations in SHR vascular p22phox expression as well as NADPH oxidase, xanthine oxidase and catalase activities; had no effect on vascular CuZn-SOD and Mn-SOD expression or total SOD activity; and reversed the elevation in SHR vascular glycated/free extracellular-SOD expression ratio and plasma glucose without changes in plasma AGEs. Conclusion: LGF treatment of SHRs normalizes the level of plasma oxidative stress biomarkers through a reduction of vascular superoxide anion produced by NADPH and xanthine oxidases. These effects might be linked to the cardiovascular regenerative actions of LGF. Keywords: NADPH oxidase, oxidative stress, SHR, superoxide anion, xanthine oxidase Abbreviations: AGEs, advanced glycation end products; LGF, liver growth factor; MDA, protein-bound malondialdehyde INTRODUCTION T here is compelling evidence that excessive reactive oxidative and nitrosative species (ROS, RNS) contribute to the pathogenesis of hypertension [1–9]. Therefore, there is great interest in the development of strategies able to reduce oxidative stress in hyperten- sion-related pathologies [10]. One of the most widely used approaches is targeting the renin–angiotensin system with angiotensin receptor ATR1 antagonists [11–13], angioten- sin-converting enzyme inhibitors [14] or combined thera- pies [15]. A second strategy is the use of small molecules to inhibit enzymatic ROS and RNS-producing systems such as NADPH oxidase [16] and xanthine oxidase [17], and a third approach is to employ ROS scavengers [18–20]. However, due to the spatial and temporal complexity of redox signaling pathways, these strategies have not always been efficient, require chronic treatments, or even had unwanted effects [21–23]. Liver growth factor (LGF) – a covalently bound albu- min–bilirubin complex identified and purified in our laboratory [24,25] – is a molecule with therapeutic potential in this field. LGF is an endogenous factor which is not detected in physiological conditions but it increases its concentration in plasma in hepato-biliary disorders, both in humans and rats [26,27]. We have previously demon- strated that short treatment (12 days) with LGF of spon- taneously hypertensive rats (SHRs) significantly reduces blood pressure, vascular fibrosis, endothelial dysfunction and left-ventricular hypertrophy [28,29]. Liver growth factor is an albumin – bilirubin complex and both molecules have been shown to be protective against oxidative stress [30,31]. Moreover, we have also demon- strated that LGF exhibits in-vitro scavenging activity against superoxide anion (O 2  ), and peroxyl and hydroxyl radicals [32]. These studies also evidenced that LGF was able to protect nitric oxide from destruction by O 2  and Journal of Hypertension 2012, 30:1185–1194 a Departamento de Fisiologı´a, Facultad de Medicina, Universidad Auto ´ noma de Madrid, b Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo-CEU and c Servicio de Bioquı´mica Experimental, Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain Correspondence to Dr M. Carmen Gonza ´ lez, Departamento de Fisiologı ´a, Facultad de Medicina, Universidad Auto ´ noma de Madrid, C/ Arzobispo Morcillo 2, 28029-Madrid, Spain. Tel: +34 914975475; fax: +34 914975478; e-mail: m.c.gonzalez@uam.es Received 8 September 2011 Revised 11 December 2011 Accepted 8 March 2012 J Hypertens 30:1185–1194 ß 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins. DOI:10.1097/HJH.0b013e328353824b Journal of Hypertension www.jhypertension.com 1185 Original Article